Method for Treating a Blood Component Containing Sample

ABSTRACT

Provided is a treatment method for damaging an erythrocyte and a leukocyte while suppressing damage to cells other than blood cells present in blood. In an embodiment, the disclosure relates to a method for treating a sample containing blood components, the method including mixing a sample containing blood components with a surfactant A, where the surfactant A is a nonionic surfactant represented by General formula R 1 —O-(EO)n-R 2  (I).

CROSS REFERENCE TO RELATED APPLICATIONS

The disclosure is based upon and claims priority from JP ApplicationSerial Nos. 2012-139051 and 2013-126403, the disclosures of which arehereby incorporated by reference herein in their entireties.

TECHNICAL FIELD

The disclosure relates to a method for treating a blood containingsample, a method for separating a rare cell and a nucleic acid in ablood containing sample, a CTC number measurement method and a reagentkit.

BACKGROUND

Although cell components of blood include an erythrocyte, a leukocyteand a blood platelet, on rare occasion, a cell other than these cellsmay exist in the blood. An example thereof is a circulating tumor cell(CTC). It is considered that metastasis of cancer occurs since a cancercell is conveyed through blood vessels or lymph vessels to any othersite in the body and proliferates there. It has been reported that thenumber of CTCs (circulating tumor cells) in the blood correlate to thepossibility of metastasis and prognosis, and thus it has been known thatthe number of CTCs in blood and a nucleic acid of a CTC are measured forproviding a guideline for prediction or decision in diagnosis,prognostication, and a therapeutic effect of cancer (in particular,metastatic cancers such as breast cancer) (see Circulating Tumor Cells;Evolving Evidence and Future Challenges, The Oncologist 2009; 14;1070-1082).

Examples of techniques for separating and detecting CTCs in bloodinclude: a method of capturing and separating CTCs in blood by use of anantibody with respect to a CTC specific surface antigen (see JP 3834326and JP 2007-178193 A); a separation method using adhesion (see WO2005/043121 and WO 2006/078994); a separation method using a densitygradient (see JP 2010-075073 A and WO 95/20429); a separation methodusing a filter (see WO 2006/116327 and WO 2008/155398); a method ofmeasuring the telomerase activity of a CTC (see WO 2010/071114); aseparation method through hemolysis by using a hypotonic solution (seeWO 2004/056978); and a method utilizing flow cytometry (see WO2008/057437). For detecting, quantifying and counting the objectseparated, measurement of a nucleic acid is generally carried out byutilizing PCR or any substance having affinity to a CTC; optical CTCmeasurements are carried out as a combination of an antigen and/or afluorescent pigment.

SUMMARY

In an embodiment, the disclosure relates to a method for treating asample containing blood components. A sample containing blood componentsand a liquid containing a surfactant A are mixed with each other. In themethod, no fixation agent is used, and the surfactant A is a nonionicsurfactant represented by General formula (I) below.

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number in the range of23 to 50, and R² is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3.

In another embodiment, the disclosure relates to a method for treating asample containing blood components. The method includes mixing a samplecontaining blood components with a liquid containing a surfactant A anda surfactant B, or with a liquid containing the surfactant A and aliquid containing the surfactant B. Here, the surfactant A is a nonionicsurfactant represented by General formula (I) below, and the surfactantB has a lytic property with respect to an erythrocyte higher than thecorresponding lytic property of the surfactant A.

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number in the range of23 to 50, and R² is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3.

In another embodiment, the disclosure relates to a method for separatingor detecting a rare cell or a nucleic acid in a sample containing bloodcomponents. Here, the method includes treating a sample containing bloodcomponents by any of the treatment methods according to the disclosure,and separating or detecting a rare cell or a nucleic acid from thetreated sample.

In another embodiment, the disclosure relates to a method for measuringthe number of CTCs or measuring a nucleic acid of a CTC in a samplecontaining blood components. Here, the method includes treating a samplecontaining blood components by the treatment methods according to thedisclosure, or separating/detecting a rare cell or a nucleic acid from asample containing blood components by the separation/detection methodsaccording to the disclosure.

In another embodiment, the disclosure relates to a method for assaying arare cell in a sample containing blood components. Here, the methodincludes treating a sample containing blood components by the treatmentmethods according to the disclosure, and subsequently assaying by amethod including an observation of movements of the cell or an activitymeasurement.

In another embodiment, the disclosure relates to a reagent kit includingthe surfactant A and/or the surfactant B to be used for the treatmentmethods according to the disclosure, the separation/detection methodsaccording to the disclosure, the methods for measuring a CTC number or aCTC nucleic acid according to the disclosure, and/or the assayingmethods according to the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between a mixing ratio of asurfactant No. 2 to a surfactant No. 1 and a survival rate of cancercells.

FIG. 2 is a graph showing the relationship between a mixing ratio of asurfactant No. 3 to a surfactant No. 1 and a survival rate of cancercells.

FIG. 3 is composed of microscopic photographs showing the result ofcultivating cancer cells collected by centrifugation separation afterthe treatment with a surfactant mixture.

FIG. 4 is composed of microscopic photographs showing filtered samples:sample (A) has not been treated with a surfactant mixture, and sample(B) has been treated with a surfactant mixture.

FIG. 5 shows the result of a fluorescence microscopic observation aftertreatment with a surfactant mixture with respect to a blood sample thatincludes cancer cells (SNU-1) stained with green fluorescence andleukocytes stained with blue fluorescence.

FIG. 6 shows the result of a Coulter type flow cytometry analysis of asample containing blood components, which includes a cancer cell (SNU-1)suspension or leukocytes: (A) has been treated with a surfactant mixturewhile (B) has not been subjected to such a treatment.

FIG. 7 shows the result of a Coulter type flow cytometry analysis of asample containing blood components, which includes cancer cells (SNU-1)and leukocytes: (A) has been treated with a surfactant mixture while (B)has not been subjected to such a treatment.

FIG. 8 is a graph showing the survival rates of samples containing bloodcomponents, which include cancer cells (SNU-1) or leukocytes suspendedin a physiological saline, after a treatment with surfactant mixtures ofvarious pHs.

DETAILED DESCRIPTION

For assaying a rare cell in a sample containing blood components or forseparating and cultivating the cell, it is necessary to eliminateefficiently the cell components in the blood (in particular,erythrocytes and leukocytes) without causing serious damage to the rarecell.

Therefore, with the foregoing in mind, the disclosure provides a simpletreatment method that enables removal of blood cells quickly whilesuppressing damage to cells other than the blood cells in the samplecontaining the target component. Further, the disclosure provides asimple treatment method, or separation/detection method that improvesthe purity of CTCs by removing blood cells and the like, therebyenabling suppression of noises at the time of counting the CTCs ormeasuring the nucleic acid, and also enabling detection of the CTCs.

In an embodiment, the disclosure relates to a method for treating asample containing blood components. A sample containing blood componentsand a liquid containing a surfactant A are mixed with each other. In themethod, no fixation agent is used, and the surfactant A is a nonionicsurfactant represented by General formula (I) below.

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number in the range of23 to 50, and R² is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3.

In another embodiment, the disclosure relates to a method for treating asample containing blood components. The method includes mixing a samplecontaining blood components with a liquid containing a surfactant A anda surfactant B, or with a liquid containing the surfactant A and aliquid containing the surfactant B. Here, the surfactant A is a nonionicsurfactant represented by General formula (I) below, and the surfactantB has a lytic property with respect to an erythrocyte higher than thecorresponding lytic property of the surfactant A.

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number in the range of23 to 50, and R² is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3.

In another embodiment, the disclosure relates to a method for treating asample containing blood components. The method includes mixing a samplecontaining blood components with either a liquid containing a surfactantA and a surfactant B or with a liquid containing the surfactant A and aliquid containing the surfactant B. Here, the surfactant A ispolyoxyetheylene octyl dodecyl ether (EO=23-50). The surfactant B is anonionic surfactant having a lytic property with respect to anerythrocyte higher than the corresponding lytic property of thesurfactant A, and it is selected from the group consisting ofpolyoxyethylene polyoxypropylene alkylether, polyoxyethylene octyldodecyl ether (EO=8-22), polyoxyethylene fatty acid ester, saccharosefatty acid ester, sorbitan fatty acid ester, and a combination thereof.

In another embodiment, the disclosure relates to a method for separatingor detecting a rare cell or a nucleic acid in a sample containing bloodcomponents. Here, the method includes treating a sample containing bloodcomponents by any of the treatment methods according to the disclosure,and separating or detecting a rare cell or a nucleic acid from thetreated sample.

In another embodiment, the disclosure relates to a method for measuringthe number of CTCs or measuring a nucleic acid of a CTC in a samplecontaining blood components. Here, the method includes treating a samplecontaining blood components by the treatment method according to thedisclosure, or separating/detecting a rare cell or a nucleic acid from asample containing blood components by the separation/detection methodaccording to the disclosure.

In another embodiment, the disclosure relates to a method for assaying arare cell in a sample containing blood components. Here, the methodincludes treating a sample containing blood components by the treatmentmethod according to the disclosure, and subsequently assaying by amethod including an observation of movements of the cell or an activitymeasurement.

In another embodiment, the disclosure relates to a reagent kit includingthe surfactant A and/or the surfactant B to be used for the treatmentmethod according to the disclosure, the separation/detection methodaccording to the disclosure, the method for measuring a CTC number or aCTC nucleic acid according to the disclosure, and/or the assaying methodaccording to the disclosure.

According to the disclosure, it is possible to lyse erythrocytes andcause damage to leukocytes while suppressing damage to any cells otherthan the blood cells present in the sample containing the blood.

It is expected that an assay of rare cells such as a circulating tumorcells in blood will be increased further and thus there has been ademand for a simpler method that causes fewer losses. In general, tensof billions of erythrocytes and tens of millions of leukocytes areincluded in 10 ml of blood, while the number of CTCs in the same bloodis as small as 0 to several thousands. For this reason, many technicalproblems exist in a direct analysis of CTCs. For example, treatmentssuch as separation and concentration are required. Therefore, there hasbeen a demand for a technique of concentrating and separating CTCs fromblood more efficiently and/or more easily.

As a separation method utilizing an antigen-antibody reaction, there hasbeen proposed a method of concentrating cells from blood by usingmagnetic beads to which an antibody has been applied and a magneticfield (see JP 3834326 and JP 2007-178193 A). However, since overcrowdingblood cells that can hinder the contact of the antibody to target cellsare present in the blood, it takes time for the reaction and collectionof cells. Furthermore, due to the presence of the blood cells, thereaction volume is large and the amount of reagent per volume isincreased, and thus the cost is raised. Furthermore, in a case ofconcentrating by using the beads, many blood cells are caught and thusthe purity is degraded to create a false-positive. Furthermore, when acleaning process for eliminating the blood cells is repeated, the cellsof the target may be lost.

In a separation method utilizing adhesion (see WO 2005/043121 and WO2006/078994), blood cells and blood platelets deposit on the bottom faceof a typical carrier or a laboratory dish for cell cultivation. However,cancer cells cannot adhere to the laboratory dish and thus it isimpossible to separate out the cancer cells. For solving this problem,proposed is a method of fixing a substance that has high affinity (suchas an antibody) to the laboratory dish or to the carrier so as to allowthe cells to adhere thereto. Similarly however, while there is anecessity that the cells and the substance with high affinity getcontact with each other on the bottom face, the blood cells may hinderthe contact. As a result, for increasing the chances of contact, it isrequired to devise a modification in the structure of the carrier or amethod of stirring, and thus increasing the complexity of the processes.

In a centrifugation separation method, layers of the blood cells, namelya layer of erythrocytes and a layer of leukocytes in this order from thebottom, are formed after the centrifugation. Since many cancer cellshave an approximate density and size to those of the leukocytes, manyleukocytes are mixed with the cancer cells. Namely, the centrifugationseparation is inferior in purity, resulting in a false-positive. Inaddition to that, the respective layers are located to be so proximateto each other that many erythrocytes are mixed in to degrade thecollection rate and the purity.

Separation by a density gradient centrifugation method (JP 2010-075073 Aand WO 95/20429) is used for various purposes as a method of collectingcells from the blood. However, in such a separation utilizing thedensity of the cells, since the size and density of the cancer cells areapproximate to those of mononuclear cells of the leukocytes, manymononuclear cells are mixed in to cause the false-positive. Further inthe density gradient centrifugation separation, the processes tend to becomplicated. For example, it is required to collect the cells' stratawithout disturbing the liquid after the separation so that therespective strata will not be mixed with each other.

In a separation using a filter (WO 2006/116327 and WO 2008/155398), thedifference in size of the rare cells and the deformability of the bloodcells are used for separating the blood cells and the cancer cells fromeach other. While many erythrocytes and many leukocytes pass through thefilter, a part of erythrocytes and a part of leukocytes remain on themembrane. When the pore size of the membrane is smaller than 8 μm,clogging will occur easily. In contrast, when the filtration pressure isincreased to improve the efficiency in passing the blood cells throughthe filter, the flow rate is increased to damage the rare cells. In somecases, the rare cells may pass the pores and thus the collection ratedeteriorates.

In a flow cytometry, since several millions of erythrocytes and severalthousands to about ten thousands of leukocytes are present in 1 μL ofblood, generally it is impossible to count the number directly.Therefore, it is required to add a hemolytic agent for eliminating theerythrocytes and to dilute the solution for detecting the respectivecells at the time of measurement. It is extremely difficult to dilutethe 10 mL of blood, to count the respective cells among the tens ofbillions of erythrocytes and hundreds of millions to several thousandsof leukocytes, and to separate and count the cancer cells. In a case ofan electrical resistance system, since there are leukocytes assubstantially large as the cancer cells, many leukocytes are mixed inafter the separation so as to cause a false-positive.

As mentioned above, the conventional separation-condensation techniquesare regarded as insufficient to detect and count the rare cells withregard to a required sensitivity, efficiency and specificity, due to thesurvival of blood cells. If the blood cells in a sample can be treatedby separation, elimination, removal or the like, in a separation usingan antigen-antibody reaction, it is possible to subject the sample tocentrifugation after lysis so as to allow the sample to react with asmall volume of re-suspended solution, thereby shortening the reactiontime and reducing the cost for the reagent. Similarly, in a separationthat utilizes adhesion, cultivation and adhesion-separation becomeavailable by use of a laboratory dish for cultivation. Further in aseparation that uses centrifugation-separation and density gradientcentrifugation, the rare cells are collected easily. Moreover, in a caseof using a filter, the analysis is carried out easily. That is, bytreating the blood cells in the sample containing the blood, the errorfactor in counting and quantifying the rare cells (false-positive) isdecreased so that quick measurement is realized.

Further, if the rare cells in the sample containing the blood can beassayed alive or can be separated and cultivated, it is possible toassay and analyze the rare cells more precisely. In general however,saponin and triton that are the nonionic surfactants utilized ashemolytic reagents for blood cell elimination, also damage rare cellssuch as the cancer cells so as to cause the cells to die, therebydestroying the cells. Therefore in the case of analyzing the target cellby using the surfactant, as mentioned in WO 2010/071114, the rare cellsin the sample containing the blood are fixed in advance with acrosslinking reagent (a fixation reagent) such as formaldehyde andparaformaldehyde and subsequently allowed to react with the hemolyticreagent. The crosslinking agent such as formaldehyde andparaformaldehyde typically forms an intermolecular crosslink via freeamino groups so as to maintain the cell membrane and the cellularstructure. However, in return for the effect of maintaining the cellularstructure, the fixation operation makes it difficult to conduct aseparation-cultivation, observation, activity measurement, furtheranalysis of pharmacometrics or the like for the living rare cells.

Moreover, the surfactant at the concentration recited in WO 2010/071114cannot lyse the blood directly, and may even damage the cancer cells.Further, many leukocytes survive.

Formaldehyde and paraformaldehyde are fixation agents to be compatiblyused typically in the case of using a hemolytic reagent. As theformaldehyde and the paraformaldehyde are powerful medicines, they mayaffect the health of the user. Although WO 2010/071114 and WO2004/056978 describe a method of eliminating blood cells by use ofammonium chloride salt and/or a hypotonic solution, the dilution ratiois greater and thus a larger reaction tank is needed. Moreover, sincethe substance does not have lytic power as much as the surfactant,aggregates of cell fragments or the like will be formed easily.

The disclosure is based on the knowledge that in one or moreembodiments, a surfactant A having a low lytic property with respect toerythrocytes and causing less damage to cells is used to lyse theerythrocytes while suppressing damage to rare cells present in theblood. Further, the disclosure is based on a knowledge that in one ormore embodiments, a surfactant B having a powerful lytic property withrespect to erythrocytes and causing more damage to rare cells and asurfactant A having low lytic property with respect to erythrocytes andcausing less damage to rare cells are combined to treat the bloodsample, so that it is possible to suppress damage to rare cells in theblood while lysing the erythrocytes and damaging the leukocytes. Anotheradvantage is that these treatments can be carried out at a low cost.

Although the detail of the mechanism for enabling removal of the bloodcells while suppressing damage to the rare cells in the blood by thesurfactant A has not yet been clarified, the following inferences can bemade. With regard to the cells, it is considered that differences in thecytoskeleton and the membrane are involved. Since the blood cells thatcirculate in vivo pass through the capillary vessels and the like, thecytoskeleton may deform easily. Further, the phospholipid bilayer of thecell membrane has a high flowability while the interaction between thehydrophobic parts of the phospholipid is weak. As a result, such a bloodcell will be easily destroyed when it is subjected to the action of thesurfactant. On the other hand, it is considered that the rare cells arerarely destroyed because specific filaments such as cytokeratin isspread across the interior of the cell and bonded to the cell membraneor the like, and the phospholipid bilayer is less flowable in comparisonwith the blood cells. Further, with regard to the surfactant, it isconsidered that the surfactant interacts with the protein, cholesteroland phospholipid on the cell membrane and lyses the cell. For thesurfactant A, it is preferable that the molecular structure has abranched chain similar to the structure of a hydrophobic group of aphospholipid. The influence of the lytic power and the steric hindrancecaused by the hydrophilic group is controlled by balancing the number ofthe added hydrophobic groups and hydrophilic groups, for example, toachieve lysing the blood cells without destroying the rare cells.However, it should be noted that the disclosure is not limited to thesemechanisms.

Although the detail of the mechanism for enabling removal of blood cellswhile suppressing damage to the rare cells in the blood by thecombination of the surfactants A and B has not been clarified yet, thefollowing inference can be made. By combining the surfactant B having ahigh lytic property to the blood cells with the surfactant A, thesurfactants A and B form a mixed micelle. This mixed micelle at acertain rate acts selectively on the blood cells without hindering theselectivity of the surfactant A with respect to the blood cells, and thesurfactant B contacts with the blood cells and lyses blood cells rapidlywith its high lytic property. Namely, it is considered that since theblood cells can be removed quickly, the damage to the cancer cells canbe suppressed further. However, it should be noted that the disclosureis not limited to these mechanisms.

According to the treatment method of the disclosure, the blood cells canbe removed easily while suppressing damage to the cells other than theblood cells in the sample containing the blood. Therefore, in one ormore embodiments, by conducting the treatment method of the disclosure,it is possible to easily carry out the separation, condensation,measurement and/or cultivation and assay of rare cells in the blood, inparticular CTCs (circulating tumor cells). Even if a leukocyte isstained in a case of staining and labeling the CTC, the leukocytebecomes a dead cell or it is damaged after the treatment according tothe disclosure. Thus, the staining substance or the like leaks out andthe signal (noise) from the leukocyte is lowered. Therefore, even in anonspecific staining method that utilizes a fluorescent materialaccumulated within a cell or a material to be converted into afluorescent material through metabolism (e.g., CellTracker (GreenCMFDA/C7025 Invitrogen)), in one or more embodiments, a false-positivein detection, measurement, observation or the like of CTCs can besuppressed by conducting the treatment method of the disclosure.

[Sample Containing Blood Components]

In one or more embodiments, “a sample containing blood components” canbe described also as “a blood-components-containing sample”, whichindicates a sample to which the treatment method of the disclosure canbe applied, and the examples include blood, a blood-derived substanceincluding an erythrocyte component, a bodily fluid mixed with blood or ablood-derived substance, and a sample prepared therefrom. In one or moreembodiments, the “bodily fluid” may include blood, lymph, ascites,pleural fluid, cerebrospinal fluid, saliva, urine, and breast milk. Anexample of the blood is blood collected from a living body, and examplesof the living body include the human being and animals other than thehuman being (e.g., mammals). Examples of the blood-derived substancecontaining erythrocyte components include a substance that is separatedor prepared from blood and that contains an erythrocyte component or thedilution/concentrate thereof. The examples include a blood cell fractionfrom which blood plasma has been excluded, a blood cell concentrate, afreeze-dried substance of blood or blood cell, a sample prepared bysubjecting whole blood to hemolysis, centrifuged blood, spontaneouslysedimented blood, washed blood cells, specific fractions and the like.Among them, in one or plural unlimited embodiment, from the viewpoint ofeasy and rapid treatment and from a viewpoint of suppression of damageto rare cells in the blood, blood or a blood-derived substance(s) isused preferably for the blood containing sample.

In one or more embodiments, “a rare cell in blood” or “a rare cell in asample containing blood components” indicates a cell other than acellular component that can be contained in the blood of a human beingor an animal other than a human being (e.g., an erythrocyte, leukocyte,and blood platelet), which includes a tumor cell and/or a cancer cell.In general, a tumor cell or a cancer cell that circulates in blood iscalled a CTC. The number of these rare cells in blood is in general inthe range of 0 to several thousands in 10 ml of blood. In one or moreembodiments, the “rare cell in blood” or the “rare cell in a samplecontaining blood components” indicates a cell selected from the groupconsisting of a cancer cell, a circular tumor cell, a vascularendothelial cell, a vascular endothelial precursor cell, a cancer stemcell, an epithelial cell, a hematopoietic stem cell, a mesenchymal cell,a fetal cell, and a combination thereof. In the present invention, inone or more embodiments, a “a nucleic acid” to be separated or detectedis a nucleic acid selected from the group consisting of: RNA and DNA ina blood component; RNA and DNA in a cancer cell, a circular tumor cell,a vascular endothelial cell, a vascular endothelial precursor cell, acancer stem cell, an epithelial cell, a hematopoietic stem cell, amesenchymal cell and a fetal cell; and a combination thereof.

In one or more embodiments, a “removal of blood cell” indicateserythrocyte hemolysis and/or leukocyte growth inhibition. For instance,the erythrocyte hemolysis and the leukocyte growth inhibition can bechecked by the method in Examples. In one or more embodiments,“leukocyte growth inhibition” may include death of a leukocyte(degradation of survival rate), hemolysis, and/or inhibition ofleukocyte proliferation. In one or more embodiments, hemolysis of anerythrocyte can be observed by measuring the change in turbidity in ablood containing sample. In one or more embodiments, the degree ofturbidity fluctuates a little depending on the size of the measurementcell, the standard for hemolysis may be set to reduction of the degreeof turbidity to a half or less from the initial turbidity. Specifically,30 μl of a blood containing sample and 30 μl of a reagent are mixed,which can be checked through measurement by use of a micro-plate reader.For example, in a case of a solution prepared by mixing blood with PBSor distilled water under the above-mentioned conditions, the initialturbudity is 2 to 1.5 as an absorbance value at a wavelength of 650 nm.Turbidity lowered to 0.8 or less, 0.6 or less, or 0.5 or less indicatesthe occurrence of hemolysis. Alternatively, in one or more embodiments,survival rate of a leukocyte in a blood containing sample which is 50%or less, less than 50%, 40% or less, less than 40%, or even 30% or lessindicates occurrence of leukocyte growth inhibition.

In one or more embodiments, a liquid containing the surfactant A has theblood cell elimination effect as mentioned in the following (1).

(1) A control sample is prepared by diluting a predetermined amount ofblood with distilled water so that the absorbance value measured byabsorption photometry by use of a micro-plate reader and light having awavelength of 600 nm or more will be not less than 1 and less than 2;

a test sample is prepared by replacing the distilled water for thecontrol sample by a liquid containing 2.5 to 5 w/v % of the surfactantA;

where the absorbance value of the test sample measured by the absorptionphotometry by use of light having a wavelength of 600 nm or more isdecreased to a half the absorbance value of the control sample withinone hour.

In one or more embodiments, regarding the blood cell elimination effectof the above (1), in the liquid containing the surfactant A, theabsorbance value of the test sample measured by the absorptionphotometry using the light having a wavelength of 600 nm or more isdecreased to a half the absorbance value of the control sample within 60minutes; within 40 minutes, or within 30 minutes.

In one or more embodiments, the liquid containing both the surfactant Aand the surfactant B, or a liquid containing the surfactant A and aliquid containing the surfactant B or a mixture of the two liquids hasthe blood cell elimination effect as mentioned in the following (2).

(2) A control sample is prepared by diluting a predetermined amount ofblood with distilled water so that the absorbance value measured byabsorption photometry by use of a micro-plate reader and light having awavelength of 600 nm or more will be 1 or more and less than 2;

a test sample is prepared by replacing the distilled water for thecontrol sample by a liquid containing 2.5 to 5 w/v % of the surfactant Aand the surfactant B;

where the absorbance value of the test sample measured by the absorptionphotometry by use of light having a wavelength of 600 nm or more isdecreased to a half the absorbance value of the control sample within0.5 hours.

In one or more embodiments, regarding the blood cell elimination effectof the above (2), in the liquid containing both the surfactant A and thesurfactant B, a liquid containing the surfactant A and a liquidcontaining the surfactant B or a mixture of the two liquids, theabsorbance value of the test sample measured by the absorptionphotometry using the light having a wavelength of 600 nm or more isdecreased to half of the absorbance value of the control sample within30 minutes; within 20 minutes, or within 15 minutes.

Regarding the test sample for assessing the blood cell eliminationeffect, the concentration of the surfactant A in the liquid containingthe surfactant A is for example in a range of 0.001 w/v % to 10 w/v %.The total concentration of the surfactant A and the surfactant B in theliquid containing both the surfactant A and the surfactant B is forexample in a range of 0.001 w/v % to 10 w/v %.

In one or more embodiments, “a combination thereof” of a group composedof number n of components may include a combination of 2 to n componentsin the group.

[Surfactant A]

The surfactant A to be used in the treatment method of the disclosure isa nonionic surfactant represented by General formula (I) below. Thesurfactant A is used alone or combined with a surfactant B having ahigher lytic property with respect to an erythrocyte, therebysuppressing damage to the rare cells in the blood caused by thesurfactant B while maintaining damage to the erythrocytes and theleukocytes. From the viewpoint of shortening the treatment time andfurther suppressing the damage to the rare cells, it is preferable thatthe surfactant A is combined with the surfactant B (described below)having a higher lytic property with respect to an erythrocyte.

R¹—O-(EO)n-R²  (I)

In the formula (I) above, R¹ is a hydrocarbon group having a branchedchain and having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number in the range of23 to 50, and R² is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3.

From the viewpoint of removing the blood cells while suppressing damageto the rare cells in the blood, R¹ in the formula (I) is a hydrocarbongroup having a branched chain and having a carbon number in the range of12 to 40, and preferably an alkyl group having a branched chain andhaving a carbon number in the range of 12 to 30. In one or moreembodiments, from the viewpoint of removing the blood cells whilesuppressing damage to the rare cells in the blood, the carbon number ofthe hydrocarbon group and the alkyl group is preferably in the range of12 to 28, more preferably 14 to 26, further preferably 16 to 24, stillfurther preferably 18 to 22, and still further preferably 20. Preferredexamples for R¹ in the formula (I) include: an isohexyl group, anisononyl group, an isodecyl group, an isododecyl group, an isohexadecylgroup, a hexyldecyl group, a hexyldodecyl group, a hexylhexadecyl group,an octyldecyl group, an octyldodecyl group, an octylhexadecyl group, anonyldecyl group, a nonyldodecyl group, and a nonylhexadecyl group; andfrom the similar viewpoint, an octyldodecyl group is preferred. R² inthe formula (I) is a hydrogen atom or a hydrocarbon group having acarbon number in the range of 1 to 3. In one or more embodiments, fromthe viewpoint of removing the blood cells while suppressing damage tothe rare cells in the blood, it is a hydrogen atom or an alkyl grouphaving a carbon number in the range of 1 to 3. In one or moreembodiments, the alkyl group is a methyl group, an ethyl group, a propylgroup or an isopropyl group. For the above formula (I), from theviewpoint of removing the blood cells while suppressing damage to therare cells in the blood, a compound of formula (II) below is furtherpreferred.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood, theaverage addition mole number n of EO in the formulae (I) and (II) is inthe range of 23 to 50, preferably 24 or more, and more preferably 25 ormore. From the similar viewpoint, the upper limit for n is preferably 45or less, more preferably 40 or less, further preferably 30 or less, andstill further preferably 28 or less.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood, thesurfactant A is a nonionic surfactant represented by Chemical formula(I) below.

In one or more embodiments, the surfactant A to be used in the treatmentmethod of the disclosure is in the form of a liquid containing thesurfactant A or a liquid containing the surfactants A and B. In one ormore embodiments, the liquid containing the surfactant A or a liquidcontaining the surfactants A and B is an aqueous solution containing thesurfactant A or an aqueous solution containing the surfactants A and B.For the water, distilled water, ion-exchange water, ultrapure water orthe like can be used. In one or more embodiments, for the aqueoussolution, a buffer and/or pH that does not lower the survival rate ofthe rare cells may be selected.

[Surfactant B]

The surfactant B to be used in the treatment method of the disclosure isa nonionic surfactant whose power to lyse erythrocytes is higher thanthat of the surfactant A. In one or more embodiments, “lysis oferythrocyte” indicates a phenomenon of hemolysis in a case of mixing thesurfactant with a sample containing an erythrocyte. For example, in acase of treatment at the same concentration and/or the same mixingratio, when the time for hemolysis is shorter, it is regarded that thepower to lyse the erythrocyte is higher. Hemolysis is a phenomenon inwhich the cell membrane of an erythrocyte is damaged or dissolved due tovarious factors such as physical, chemical and biological factors, andhemoglobin or the like leaks out of the cell, resulting in the death ofthe erythrocyte. In one or more embodiments, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, it is preferable that the surfactant B to be used in thetreatment method of the disclosure has a power of inhibiting the growthof the leukocyte which is higher than that of the surfactant A.

From the viewpoint of removing the blood cells while suppressing damageto the rare cells in the blood, the surfactant B is a nonionicsurfactant. In one or more embodiments, the examples include nonionicsurfactants selected from the group consisting of polyoxyethylenepolyoxyalkylene alkylether, polyoxyethylene alkylene ether (EO=13-22),polyoxyethylene fatty acid ester, saccharose fatty acid ester, sorbitanfatty acid ester, and a combination thereof.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably the surfactant B of polyoxyethylene polyoxyalkylenealkylether type is a nonionic surfactant represented by a formula (III)below.

R³—O-(AO)m/(EO)n-R⁴  (III)

In the formula (III) above, R³ is an alkyl group having a carbon numberin the range of 1 to 24, R⁴ is a hydrogen atom or an alkyl group havinga carbon number in the range of 1 to 3, AO is an oxyalkylene grouphaving a carbon number in the range of 3 to 6, EO is an oxyethylenegroup, m and n are average addition mole numbers of AO and EOrespectively, where m is a number in the range of 1 to 100 and n is anumber in the range of 0 to 50, and “/” indicates that the AO group andthe EO group may be added at random or as a block regardless of theorder.

In one or more embodiments, examples of R³ in the formula (III) includemethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl),pentadecyl, hexadecyl(cetyl), heptadecyl, octadecyl (stearyl),nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyland the like. In the formula (III) above, R⁴ is a hydrogen atom or ahydrocarbon group having a carbon number in the range of 1 to 3. In oneor more embodiments, from the viewpoint of removing the blood cellswhile suppressing damage to the rare cells in the blood, it is ahydrogen atom or an alkyl group having a carbon number in the range of 1to 3, and in one or more embodiments, the alkyl group is a methyl group,an ethyl group, a propyl group or an isopropyl group. In one or moreembodiments, examples of the AO in the formula (III) includepolyoxypropylene, polyoxybutylene and the like. In one or moreembodiments, from the viewpoint of removing the blood cells whilesuppressing damage to the rare cells in the blood, m in the formula(III) is preferably in the range of 1 to 60, and more preferably 1 to30. Similarly, n is preferably in the range of 1 to 50, and morepreferably 1 to 25.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably the surfactant B of the formula (III) is a polyoxyethylenepolyoxypropylene alkylether. In one or more embodiments, from theviewpoint of removing the blood cells while suppressing damage to therare cells in the blood, the polyoxyethylene polyoxypropylene alkyletherhas a HLB value (hydrophilic-lipophilic balance value) in the range of10.0 to 18.0, more preferably 12.0 to 15.0, and further preferably 12.5to 13.5. From a similar viewpoint, preferably the polyoxyethylenepolyoxypropylene alkylether has a cloudy point (° C./2% aqueoussolution) in the range of 34° C. to 88° C., more preferably 40° C. to75° C., and further preferably 50° C. to 60° C.

In one or more embodiments, examples of commercial products for thesurfactant B of the formula (III) include: EMALEX DAPE-207, EMALEXDAPE-210, EMALEX DAPE-212, EMALEX DAPE-215, EMALEX DAPE-220, EMALEXDAPE-230 (all of which are manufactured by Nihon Emulsion Co., Ltd.);Plurafac (trade name) LF300, Plurafac (trade name) LF400, Plurafac(trade name) LF431, Plurafac (trade name) LF711, Plurafac (trade name)LF900, Plurafac (trade name) LF901, and Plurafac (trade name) LF1300(all of which are manufactured by BASF Japan Ltd.); EMULGEN LS-106,EMULGEN LS-110, EMULGEN LS114, and EMULGEN MS110 (all of which aremanufactured by Kao Corporation); WONDERSURF RL-80, WONDERSURF RL-100,WONDERSURF RL-140, WONDERSURF S-800, WONDERSURF 5-1000, and WONDERSURFS-1400 (all of which are manufactured by AOKI OIL INDUSTRIAL CO., LTD.);and UNISAFE PKA-5015, UNISAFE PKA-5016, UNISAFE PKA-5017, and UNISAFELM-2 (all of which are manufactured by NOF CORPORATION).

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably the surfactant B of polyoxyethylene alkylether type(EO=13-22) is a nonionic surfactant represented by the formula (IV)below.

R⁵—O-(EO)n-R⁶  (IV)

In the formula (IV) above, R⁵ is a hydrocarbon group having a carbonnumber in the range of 12 to 40, EO is an oxyethylene group, n is anaverage addition mole number of EO in the range of 8 to 22, and R⁶ is ahydrogen atom or a hydrocarbon group having a carbon number in the rangeof 1 to 3.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably R⁵ is a hydrocarbon group having a branched chain and havinga carbon number in the range of 12 to 40, more preferably an alkyl grouphaving a branched chain and having a carbon number in the range of 12 to30. In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably the carbon number of the hydrocarbon group and the alkylgroup is in the range of 12 to 28, more preferably 14 to 26, furtherpreferably 16 to 24, still further preferably 18 to 22, and stillfurther preferably 20. Preferred examples for R⁵ of the formula (IV)include an isohexyl group, an isononyl group, an isodecyl group, anisododecyl group, an isohexadecyl group, a hexyldecyl group, ahexyldodecyl group, a hexylhexadecyl group, an octyldecyl group, anoctyldodecyl group, an octylhexadecyl group, a nonyldecyl group, anonyldodecyl group, and a nonylhexadecyl group; and from the similarviewpoint, an octyldodecyl group is preferred. R⁶ in the formula (IV) isa hydrogen atom or a hydrocarbon group having a carbon number in therange of 1 to 3. In one or more embodiments, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, it is a hydrogen atom or an alkyl group having a carbonnumber in the range of 1 to 3, and in one or more embodiments, the alkylgroup is a methyl group, an ethyl group, a propyl group or an isopropylgroup. For the formula (IV), from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood, a formula(V) below is further preferred.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood,preferably the average addition mole number n of the EO in the formulae(IV) and (V) is in the range of 10 to 22, more preferably 13 to 22,further preferably 15 to 21, still further preferably 18 to 21, andstill further preferably 20.

In one or more embodiments, the surfactant B of polyoxyethylene fattyacid ester type is a nonionic surfactant represented by a formula (VI)below.

R⁷—COO-(EO)n-R⁸  (VI)

In the formula (VI) above, R⁷ is a hydrocarbon group having a carbonnumber in the range of 10 to 40, EO is an oxyethylene group, n is anaverage addition mole number of EO in the range of 6 to 160, and R⁸ is ahydrogen atom or a hydrocarbon group having a carbon number in the rangeof 1 to 3.

From the viewpoint of removing the blood cells while suppressing damageto the rare cells in the blood, R⁷ in the above formula (VI) is ahydrocarbon group having a carbon number in the range of 10 to 40. Inone or more embodiments, it is an alkyl group having a carbon number inthe range of 10 to 30. In one or more embodiments, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, the carbon number of the hydrocarbon group and the alkylgroup is in the range of 10 to 20, 10 to 18, 10 to 16, or 12. R⁸ in theabove formula (VI) is a hydrogen atom or a hydrocarbon group having acarbon number of 1 to 3. In one or more embodiments, from the viewpointof removing the blood cells while suppressing damage to the rare cellsin the blood, it is a hydrogen atom or an alkyl group having a carbonnumber in the range of 1 to 3. In one or more embodiments, the alkylgroup is a methyl group, an ethyl group, a propyl group or an isopropylgroup. In one or more embodiments, from the viewpoint of removing theblood cells while suppressing damage to the rare cells in the blood, theaverage addition mole number n of EO in the formula (VI) is in the rangeof 6 to 20, 8 to 18, to 16, or 12. In one or more embodiments, from theviewpoint of removing the blood cells while suppressing damage to therare cells in the blood, the surfactant B in the formula (VI) ispolyoxyethylene (12) monolaurate.

In one or more embodiments, the surfactant B is a nonionic surfactanthaving a sugar moiety as a hydrophilic part and either a fatty acidchain or an alkyl chain as a hydrophobic part.

There is no particular limitation on the sugar moiety and the examplesinclude sugar moieties of monosaccharide, disaccharide, oligosaccharideand the like. The number of the monosaccharides in the sugar moiety isnot limited in particular, and it may be in the range of 1 to 20 forexample. Although there is no particular limitation on the sugar moietyof the monosaccharide, the examples include glucose residue, galactoseresidue, mannose residue, thioglucose residue, arabinose residue, xyloseresidue, glucuronic acid residue, and glucosamine residue. Althoughthere is no particular limitation on the sugar moiety of thedisaccharide, the examples include sucrose residue (saccharose residue),lactose residue, maltose residue, and thiomaltose residue; preferably,sucrose residue.

There is no particular limitation on the fatty acid chain, and theexamples include fatty acid residue and an alkyl group. There is noparticular limitation on the fatty acid residue, and it may be asaturated fatty acid residue or an unsaturated fatty acid residue forexample. Examples of the fatty acid residue include, without anyparticular limitations, a linear fatty acid residue, a branched fattyacid residue, and a cyclic fatty acid residue. The carbon number of thefatty acid residue is not limited particularly, and it is in the rangeof 4 to 28 for example, and preferably in the range of 10 to 22.Although there is no particular limitation on the saturated fatty acidresidue, the examples include a capric acid residue, a lauric acidresidue, a myristic acid residue, a pentadecylic acid residue, apalmitic acid residue, a stearic acid residue, an arachidic acidresidue, and a behenic acid residue. Preferably, they are acyl groupssuch as a decanoyl group, a dodecanoyl group, a tetradecanoyl group, apentadecanoyl group, a hexadecanoyl group, an octadecanoyl group, anicosanoyl group and a docosanoyl group. Although there is no particularlimitation on the unsaturated fatty acid residue, the examples includean oleic acid residue, and a linoleic acid residue. For example, acylgroups such as a cis-9-octadecenoyl group and acis,cis-9,12-octadeoctadecadinoyl group are preferred. For the fattyacid residue, from the viewpoint of removing the blood cells whilesuppressing damage to the rare cells in the blood, the capric acidresidue and the lauric acid residue are preferred.

The alkyl group is not limited particularly. For example, it may be alinear alkyl group or a branched alkyl group. The carbon number of thealkyl group is not limited particularly, and for example, it may be inthe range of 1 to 18. Specific examples of the alkyl groups include,without any particular limitation, a methyl group, an ethyl group, an-propyl group, an isopropyl group, a n-butyl group, an isobutyl group,a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, aheptyl group, an octyl group, a nonyl group, a decyl group, an undecylgroup, a dodecyl group, a tridecyl group, a tetradecyl group, apentadecyl group, a hexadecyl group, a heptadecyl group, an octadecylgroup, an nonadecyl group, and an icosyl group.

Specific examples of the nonionic surfactant having a sugar moietyinclude, without any particular limitation, saccharose (sucrose) fattyacid ester, alkylglucoside, and alkyl oligosaccharide.

Although there is no particular limitation on the saccharose (sucrose)fatty acid ester, the examples include sucrose caprate, sucrose laurate,sucrose myristate, sucrose palmitate, sucrose stearate, sucrosebehenate, sucrose oleate, and sucrose linoleate; preferably, sucrosecaprate and sucrose laurate. There is no particular limitation on thesaccharose (sucrose) fatty acid ester, and the examples includemonoester, diester, and triester. Although there is no particularlimitation on the saccharose fatty acid monoester, the examples includesucrose monocaprate, sucrose monolaurate, sucrose monomyristate, sucrosemonopalmitate, sucrose monostearate, sucrose monobehenate, sucrosemonooleate, and sucrose monolinoleate; preferably, sucrose monocaprateand sucrose monolaurate. Although there is no particular limitation onthe saccharose fatty acid diester, the examples include sucrosedicaprate, sucrose dilaurate, sucrose dimyristate, sucrose dipalmitate,sucrose distearate, sucrose dibehenate, sucrose dioleate, and sucrosedilinoleate; preferably, sucrose dicaprate and sucrose diaurate.Although there is no particular limitation on the saccharose fatty acidtriester, the examples include sucrose tricaprate, sucrose trilaurate,sucrose trimyristate, sucrose tripalmitate, sucrose tristearate, sucrosetribehenate, sucrose trioleate, and sucrose trilinoleate; preferably,sucrose tricaprate and sucrose triaurate.

The saccharose fatty acid ester may be any of the monoester, thediester, the triester and the like, or it may be a mixture thereof.Preferably the saccharose fatty acid ester contains the monoester forexample. Although there is no particular limitation on the content ofthe saccharose fatty acid monoester, it is in the range of 50 to 100% byweight for example, and preferably in the range of 70 to 100% by weight.

Although there is no particular limitation on the alkylglucoside, theexamples include n-octyl-β-D-glucoside, n-dodecyl-β-maltoside,n-decyl-β-maltoside, n-octyl-β-D-maltoside, 3-oxamidecyl-α-D-mannoside,n-heptyl-β-thioglucoside, n-nonyl-β-D-thiomaltoside, andn-octyl-β-D-thioglucoside.

In one or more embodiments, the surfactant B to be used in the treatmentmethod of the disclosure is provided in a form of a liquid containingthe surfactant B or a liquid containing the surfactants A and B. In oneor more embodiments, the liquid containing the surfactant B or theliquid containing the surfactants A and B is an aqueous solutioncontaining the surfactant B or an aqueous solution containing thesurfactants A and B. For the water, distilled water, ion-exchange water,ultrapure water or the like can be used. In one or more embodiments, forthe aqueous solution, a buffer and/or pH that does not lower thesurvival rate of the rare cells may be selected.

The nonionic surfactants A and B may be used alone or in combination oftwo or more.

[Method for Treating a Sample Containing Blood Components]

In one or more embodiments, the disclosure relates to a method fortreating a sample containing blood components. According to the method,a sample containing blood components is mixed with a surfactant A orwith the surfactants A and B. In one or more embodiments, the method fortreating a sample containing blood components according to thedisclosure is a method of lysing erythrocytes while suppressingdegradation in the survival rate of the rare cells that may be containedin the sample, and/or degrading the survival rate of leukocytes.Therefore, in the method for treating a sample containing bloodcomponents according to the disclosure, it is preferable that the rarecells are not fixed or any fixation agent that can lower the survival ofthe rare cells is not used. In the disclosure, “fixation” includesimmobilizing the structures of cells and cell organelle while keepingthe living form. Therefore, in one or more embodiments, “not fixing”includes maintaining a state capable of morphological transformationand/or suppressing any factors that may inhibit the morphologicaltransformation.

The fixation agent includes a crosslinking reagent. The crosslinkingreagent denotes a reagent capable of maintaining a cell membrane and acellular structure by forming an intermolecular crosslink via free aminogroups. Specific examples of the fixation agent include formaldehyde,paraformaldehyde, trioxane, and glutaraldehyde. Other than thecrosslinking reagent, organic solvents such as acetone and alcohols suchas methanol and ethanol may be included.

In one or more embodiments, the method of treating a sample containingblood components according to the disclosure includes mixing a samplecontaining blood components with a liquid containing a surfactant A, orwith either a liquid containing surfactants A and B or a liquidcontaining the surfactant A and a liquid containing the surfactant B. Inone or more embodiments, the method includes adding to the samplecontaining blood components an aqueous solution containing thesurfactant A or an aqueous solution containing surfactants A and B or anaqueous solution containing the surfactant A and an aqueous solutioncontaining the surfactant B, and mixing. In one or more embodiments,addition of the liquid containing the surfactant A and the liquidcontaining the surfactant B may be an embodiment of adding any one ofthe surfactants first and then adding the other; or it may be anembodiment of adding both the surfactants at the same time. Among them,in one or more embodiments of a method of mixing a sample containingblood components with the surfactants A and B, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, it is preferable that the sample containing blood componentsand the surfactants A and B are mixed quickly to form a homogeneousmixture.

In one or more embodiments, from the viewpoint of removing the bloodcells while suppressing damage to the rare cells in the blood, and fromthe viewpoint of carrying out the treatment more easily and quickly, itis preferable that the temperature for the treatment method of thedisclosure is in the range of 0 to 50° C., more preferably 4 to 40° C.,and further preferably 10 to 37° C. Preferably the temperature indicatesa temperature at the time of allowing the sample containing the bloodcomponent to react with the surfactant A or with the surfactants A andB.

In one or more embodiments, in the treatment method of the disclosure,it is preferable that after mixing the sample containing the bloodcomponent with either the surfactant A or the surfactants A and B, forremoving the blood cells, the mixture is allowed to stand or stirred fora period of time. In one or more embodiments, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, and from the viewpoint of carrying out the treatment moreeasily and quickly, preferably the predetermined period of time is 0 to60 minutes, more preferably 1 to 30 minutes, and further preferably 1 to15 minutes.

It is preferable that the concentration of the surfactant B aqueoussolution to be mixed with the sample containing the blood component isan amount to lyse erythrocytes such that in a case of mixing thesurfactant B aqueous solution alone with the sample containing the bloodcomponent, under a condition of 10 to 0.01 w/v % and at an ambienttemperature of 25° C., in a measurement with a typical micro-platereader, in a case of allowing 30 μL of the solution to react with equalparts of the sample containing the blood component, the absorbancebecomes 0.8 or less, more preferably 0.6 or less and further preferably0.5 or less at the wavelength of 650 nm.

In one or more embodiments, it is preferable that the concentration ofthe surfactant B aqueous solution before being mixed with the samplecontaining blood components is in the range of 0.01 to 10 w/v %, morepreferably 0.01 to 5 w/v %, and further preferably 0.01 to 2.5 w/v % ina case where the sample is blood, from the viewpoint of removing theblood cells while suppressing damage to the rare cells in the blood, andfrom the viewpoint of carrying out the treatment more easily andquickly. In one or more embodiments, it is preferable that the finalconcentration of the surfactant B in a case of mixing the sample withthe surfactants A and B is in the range of 0.01 to 5 w/v %, morepreferably 0.01 to 1.25 w/v %, and further preferably 0.01 to 0.5 w/v %in a case where the sample is blood, from the viewpoint of removing theblood cells while suppressing damage to the rare cells in the blood, andfrom the viewpoint of carrying out the treatment more easily andquickly. The concentrations can be adjusted suitably in a case where thesample is prepared by concentrating or diluting blood. In thedisclosure, w/v % is a unit expressing the weight (gram) of a solutedissolved in 100 ml of the solution. For example, in a case where 5 g ofa solute is dissolved in 100 ml of a solution, it is recited as 5 w/v %.

In one or more embodiments, it is preferable that the concentration ofthe surfactant A aqueous solution before being mixed with the bloodsample is in the range of 0.01 to 10 w/v %, more preferably 0.05 to 5w/v %, and further preferably 0.5 to 5 w/v % in a case where the bloodsample is blood, from the viewpoint of removing the blood cells whilesuppressing damage to the rare cells in the blood, and from theviewpoint of carrying out the treatment more easily and quickly. In oneor more embodiments, it is preferable that the final concentration ofthe surfactant A in a case of mixing the blood sample with either thesurfactant A or with the surfactants A and B is in the range of 0.01 to10 w/v %, more preferably 0.05 to 5 w/v %, and further preferably 0.5 to5 w/v % in a case where the blood sample is blood, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, and from the viewpoint of carrying out the treatment moreeasily and quickly. The concentrations can be adjusted suitably withreference to the above-mentioned concentration in a case where thesample is prepared by concentrating or diluting blood.

The quantity ratio of the surfactant A to the surfactant B both of whichare to be mixed with the sample containing blood components can beadjusted suitably from the viewpoint of removing the blood cells whilesuppressing damage to the rare cells in the blood. In one or moreembodiments, the quantity ratio of the surfactant A to the surfactant B(A/B) in the mixture is 50/50 or more for example. From the viewpoint ofsuppressing damage to the rare cells in the blood, preferably it is60/40 or more, more preferably 70/30 or more, and further preferably80/20 or more. Further in one or more embodiments, from the viewpoint ofremoving the blood cells quickly, it is preferable that the quantityratio of the surfactant A to the surfactant B (A/B) in the mixture(weight ratio) is less than 100/0, and more preferably less than 99/1.In conclusion, in one or more embodiments, from the viewpoint ofremoving the blood cells while suppressing damage to the rare cells inthe blood, the quantity ratio of the surfactant A to the surfactant B(A/B) in the mixture (weight ratio) is for example not less than 50/50and less than 100/0, preferably in the range of 60/40 to 99/1, morepreferably 70/30 to 99/1, and further preferably 80/20 to 95/5. Here,“less than 100/0” indicates that the surfactant B of more than 0% iscontained.

In one or more embodiments, from the viewpoint of suppressing damage tothe rare cells in the blood, the total concentration of the surfactant Aand the surfactant B at the time of being mixed with a sample containingblood components is preferably 10 w/v % or less, more preferably 5 w/v %or less, further preferably 2.5 w/v % or less, still further preferably1.25 w/v % or less, and still further preferably 0.75 w/v % or less.Although there is no particular lower limit of the concentration, aconcentration to remove blood cells can be set appropriately, which isfor example 0.002 w/v % or more, 0.02 w/v % or more, or 0.04 w/v % ormore, in one or more embodiments.

Depending on the form of the sample containing blood components, abuffer and an osmotic regulator can be added to the surfactant solution(which is an aqueous solution selected from the group consisting of anaqueous solution containing the surfactant A, an aqueous solutioncontaining the surfactants A and B, and a solution containing thesurfactant B; the same shall apply hereinafter). For example, in a caseof separating rare cells from blood and subsequently allowing the rarecells to react with the surfactant solution for the purpose ofeliminating any mixed blood cells, any known buffer and/or osmoticregulator can be added to the surfactant solution as long as the cancercells are not damaged. In one or more embodiments, in the case whererare cells in blood are suspended in a solution including no serum suchas a physiological solution or a buffer solution, from the viewpoint ofsuppressing damage to the rare cells present in the blood, the pH of areaction solution after mixing therein the surfactant solution ispreferably 5.0 to 9.0, 5.5 to 8.4, 6.0 to 8.4, 5.5 to 8.0, 6.0 to 8.0,6.0 to 7.7, 6.0 to 7.5 or 6.0 to 7.4. In one or more embodiments, thesurfactant solution may have pH of 6 to 9 and the osmotic pressure of 0to 400 mOsm. In the case of a direct reaction with a sample containingblood components, in one or more embodiments, the surfactant solutioncontaining the surfactant alone may be mixed. Alternatively, the pH maybe adjusted in the above-mentioned range. The buffer is not limitedparticularly, and the examples include a phosphoric acid physiologicalbuffer solution (PBS), a citric acid buffer solution, a HEPES buffersolution, an ADA (N-(2-acetamide) iminodiacetic acid) buffer solution, aMES (2-morpholino ethane sulfonic acid) buffer solution, abis-tris(bis-(2-hydroxyethyl) imino-tris-(hydroxymethyl methane) buffersolution, a PIPES (piperazine-1,4-bis(2-ethane sulfonic acid)) buffersolution, an ACES (N-(2-acetamido)-2-aminoethane sulfonic acid) buffersolution, a MOPSO (2-hydroxy-3-morpholino propane sulfonic acid) buffersolution, a BES (N,N-bis(2-hydrozyethyl)-2-aminoethane sulfonic acid)buffer solution, and HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer solution. Examples of the osmotic regulatorinclude ethylene glycol and sodium chloride. A nucleic acid, which hasbeen obtained from broken leukocytes may be measured, decomposed, and/oreliminated by using any known technique.

[Method for Separating or Detecting a Rare Cell or a Nucleic Acid inBlood]

In the sample containing blood components after the treatment of thedisclosure, the erythrocytes are lysed and growth of the leukocytes isinhibited while damage to the rare cells (if such cells exist) in theblood is suppressed. The rare cells or the nucleic acids may beseparated or detected from the sample in this state. Therefore, thedisclosure in its aspect relates to a method for separating or detectingrare cells in blood, and the method includes treating a samplecontaining blood components by the treatment method of the disclosure,and separating or detecting the rare cells or the nucleic acids from thetreated sample containing the blood components.

In one or more embodiments, separation or detection of the rare cellscan be carried out by using a method for example separation by use ofaffinity, density separation, centrifugation separation, adhesionseparation, sizing separation, flow cytometry, electrical separation,magnetic separation, sowing on a medium, and a combination thereof.Before the separation, if necessary, the sample containing the bloodcomponent that has been treated according to the method of thedisclosure may be diluted to lower the viscosity and/or the density ofthe solution. In one or more embodiments, the separation/detectionmethod of the disclosure may include isolating the rare cells and/orconcentrating the rare cells.

Further, in one or more embodiments, the disclosure relates to a methodfor separating or detecting rare cells or nucleic acids in a samplecontaining blood components, and the method includes separation of rarecells or fractions including a nucleic acid from a sample containingblood components, and, treating the separated fractions by the method ofthe disclosure, thereby separating or detecting the rare cells or thenucleic acids from the treated fractions.

In one or more embodiments, in an example of the separation method usingaffinity, the rare cells are separated by using a solid phase (e.g.,beads or a plate) where an antibody with respect to the surface antigenof the rare cells or a probe has been immobilized. In one or moreembodiments, in an example of the separation method using adhesion, therare cells are separated by using a solid phase (e.g., beads or a plate)where molecules to adhere to the surface adhesive molecules of the rarecells have been immobilized. In one or more embodiments, an example ofthe separation method utilizing density includes a separation dependanton density of the rare cells by any of various density gradientcentrifugations. In one or more embodiments, an example of thecentrifugation separation includes a separation bycentrifugation-precipitation of the rare cells. In one or moreembodiments, the sizing separation includes separation of rare cells byfiltration using a filter having an appropriate pore size. In one ormore embodiments, the flow cytometry includes fluorescent labelingand/or immunostaining surface markers of rare cells and separating withreference to the signal. Furthermore, in one or more embodiments, it ispossible to cultivate on a medium a sample containing blood componentsafter being subjected to the treatment of the disclosure and to selectany growing cells. The rare cells separated in this manner can be usedfor an assay or a further cultivation. Alternatively in one or moreembodiments, the thus separated rare cells can be separated further onthe basis of the electrical or magnetic characteristics. By using anydetection method suitable for these separation methods, the rare cellsor the nucleic acids in the sample containing blood components can bedetected. In one or more embodiments, examples of the detection methodinclude a detection method utilizing a radioactive substance, adetection method utilizing a luminous phenomenon, a detection methodutilizing a pigment, a detection method utilizing magnetism, an electricdetection method, an optical detection method, and detection methodincluding labeling by use of the labeling method associated with adetection method selected from the group consisting of these methods anda combination thereof.

In one or more embodiments of the disclosure, it is also possible tocarry out the treatment of the disclosure and/or the separation ordetection of the disclosure with respect to the sample containing theblood components that has been subjected to the treatment of thedisclosure and/or the fractions containing the rare cells separated bythe treatment of the conventional technique or fractions from which therare cells have been removed. As a result of these treatments, it ispossible to improve the collection rate of the rare cells, or to improvethe purity of the rare cells.

[Labeling Treatment with Respect to Rare Cells in Blood]

In the treatment method of the disclosure, labeling with respect to therare cells in blood may be carried out concurrently. Labeling of rarecells is effective in the above-mentioned separation/detection method ofthe disclosure and also in the CTC number measurement method mentionedbelow, and furthermore it may be effective in assay of rare cells afterseparation. Therefore, in one or more embodiments, the treatment methodof the disclosure includes a labeling treatment. Further, labeling ofrare cells is effective in the below-mentioned CTC number measurementmethod, and also may be effective in assay of rare cells afterseparation. Therefore, in one or more embodiments, theseparation/detection method of the disclosure includes a labelingtreatment.

The labeling treatment can be carried out for example by allowing aknown labeling reagent to contact with the rare cells. Typically, it canbe carried out by mixing the labeling reagent with a sample containingblood components. The timing for mixing the labeling reagent in thesample containing the blood components may be before, simultaneously orafter addition of the surfactants A and B or may be after separation ofthe rare cells. Among them, from the viewpoint of reducing the cost forthe reagent, preferably the labeling treatment is carried out afterseparation and concentration. Although there is no particular limitationon the labeling, in one or more embodiments, the examples includeradioactive labeling, fluorescent pigment labeling, pigment staining orlabeling, magnetic labeling, charge labeling, and a combination thereof,each of which can be carried out by use of a suitable labeling reagent.

[Method for Assaying Rare Cells in a Sample Containing Blood Components]

In any other aspect, the disclosure can relate to a method for assayingrare cells in a sample containing blood components, where the methodincludes treating a sample containing blood components by a method ofthe disclosure and then assaying by a method including observation ofmovement of the cells or activity measurement.

[CTC Number Measurement Method]

It has been reported that the number of CTCs in blood correlates withpossibility of metastasis or prognosis of cancer. And thus, it has beenknown to measure the CTC number in blood for providing a guideline ofdiagnosis, prognosis, prediction/judgment of treatment effect of cancer.According to the treatment method of the disclosure and/or theseparation/detection method of the disclosure, a separation can becarried out while suppressing damage to the CTC as a rare cell.Alternatively, the nucleic acid of a CTC may be measured. Therefore, inanother aspect, the disclosure includes treating a sample containingblood components by the treatment method of the disclosure, and/or,separating a CTC from the sample containing the blood components by theseparation/detection method of the disclosure. Counting of the CTCnumber or measurement of the nucleic acid of a CTC may be carried out atthe same time of separation by the flow cytometry after an appropriatelabeling treatment for example, or the separated cells may be counted byobservation under a microscope. Alternatively, the measurement may becarried out based on fluorimetry from the viewpoint of morphology ofcancer cells or the quantities of DNA and/or RNA.

[Reagent Kit]

In a still another aspect, the disclosure relates to a reagent kit to beused for the treatment method of the disclosure, theseparation/detection method of the disclosure, and/or the CTC numbermeasurement method of the disclosure. The reagent kit contains thesurfactant A or the surfactants A and B. In one or more embodiments, thereagent kit of the disclosure may contain further a labeling reagent forlabeling the rare cells or CTC. For the labeling reagent, any of theabove-mentioned reagents can be used. In another aspect, the reagent kitof the disclosure may include an instruction manual reciting thetreatment method of the disclosure, the separation/detection method ofthe disclosure, and/or, the CTC number measurement method of thedisclosure. For the reagent kit of the disclosure, the instructionmanual may not be packed with the reagent kit of the disclosure but besupplied on web.

Namely, the disclosure can further relate to the one or more embodimentsbelow.

[A1] A method for treating a sample containing blood components, themethod comprising mixing a sample containing blood components with asurfactant A, or mixing the sample with a liquid containing thesurfactant A, wherein from the viewpoint of suppressing damage to a rarecell, it is preferable that no fixation agent is used selectively, andwherein the surfactant A is a nonionic surfactant represented by Generalformula (I) below:

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number of EO in therange of 23 to 50, and R² is a hydrogen atom or a hydrocarbon grouphaving a carbon number in the range of 1 to 3.

[A2] A method for treating a sample containing blood components, themethod comprising mixing a sample containing blood components with asurfactant A and a surfactant B, or mixing the sample with a liquidcontaining both the surfactant A and the surfactant B or with a liquidcontaining the surfactant A and a liquid containing the surfactant B,wherein from the viewpoint of suppressing damage to a rare cell, it ispreferable that no fixation agent is used selectively, wherein thesurfactant A is a nonionic surfactant represented by General formula (I)below, and wherein the surfactant B has a lytic property with respect toan erythrocyte higher than the corresponding lytic property of thesurfactant A.

R¹—O-(EO)n-R²  (I)

In the formula (I), R¹ is a hydrocarbon group having a branched chainand having a carbon number in the range of 12 to 40, EO is anoxyethylene group, n is an average addition mole number of EO in therange of 23 to 50, and R² is a hydrogen atom or a hydrocarbon grouphaving a carbon number in the range of 1 to 3.

[A3] The method for treating a sample containing blood componentsaccording to [A1] or [A2], wherein the surfactant A is polyoxyethylenealkylene ether (EO=23-35).

[A4] The method for treating a sample containing blood componentsaccording to [A1] or [A3], wherein the surfactant B is a nonionicsurfactant selected from the group consisting of polyoxyethylenepolyoxyalkylene alkylether, polyoxyethylene alkylene ether, (EO=13-22),polyoxyethylene fatty acid ester, saccharose fatty acid ester, sorbitanfatty acid ester, and a combination thereof.

[A5] The method for treating a sample containing blood componentsaccording to any one of [A2] and [A4], wherein the surfactant B is anonionic surfactant represented by General formula (III) below.

R³—O-(AO)m/(EO)n-R⁴  (III)

In the formula (III), R³ is an alkyl group having a carbon number in therange of 1 to 24, R⁴ is a hydrogen atom or an alkyl group having acarbon number in the range of 1 to 3, AO is an oxyalkylene group havinga carbon number in the range of 3 to 6, EO is an oxyethylene group, mand n are average addition mole numbers of AO and EO respectively,specifically, m is a number in the range of 1 to 100 and n is a numberin the range of 0 to 50, and “I” indicates that the AO group and the EOgroup may be added at random or as a block regardless of the order.

[A6] The method for treating a sample containing blood componentsaccording to any one of [A2] to [A5], wherein the surfactant B is anonionic surfactant represented by General formula (IV) below.

R⁵—O-(EO)n-R⁶  (IV)

In the formula (IV), R⁵ is a hydrocarbon group having a carbon number inthe range of 12 to 40, EO is an oxyethylene group, n is an averageaddition mole number of EO in the range of 8 to 22, and R⁶ is a hydrogenatom or a hydrocarbon group having a carbon number in the range of 1 to3.

[A7] The method for treating a sample containing blood componentsaccording to any one of [A2] to [A6], wherein the surfactant B is anonionic surfactant represented by General formula (VI) below.

R⁷—COO-(EO)n-R⁸  (VI)

In the formula (VI), R⁷ is a hydrocarbon group having a carbon number inthe range of 10 to 40, EO is an oxyethylene group, n is an averageaddition mole number of EO in the range of 6 to 160, and R⁸ is ahydrogen atom or a hydrocarbon group having a carbon number in the rangeof 1 to 3.

[A8] The method for treating a sample containing blood componentsaccording to any one of [A2] to [A7], wherein the surfactant B is asurfactant having a sugar moiety as a hydrophilic part and either afatty acid chain or an alkyl chain as a hydrophobic part.

[A9] The method for treating a sample containing blood componentsaccording to any one of [A2] to [A8], wherein the surfactant B issaccharose laurate.

[A10] A method for treating a sample containing blood components, themethod comprising mixing a sample containing blood components with asurfactant A and a surfactant B, or mixing the sample with a liquidcontaining both the surfactant A and the surfactant B or with a liquidcontaining the surfactant A and a liquid containing the surfactant B,wherein from the viewpoint of suppressing damage to a rare cell, it ispreferable that no fixation agent is used selectively, wherein thesurfactant A is polyoxyetheylene octyl dodecyl ether (EO=23-50);

the surfactant B is a nonionic surfactant having a lytic property withrespect to an erythrocyte higher than the corresponding lytic propertyof the surfactant A, and selected from the group consisting ofpolyoxyethylene polyoxypropylene alkylether, polyoxyethylene octyldodecyether, (EO=8-22), polyoxyethylene fatty acid ester, saccharose fattyacid ester, sorbitan fatty acid ester, and a combination thereof.

[A11] The method for treating a sample containing blood componentsaccording to any one of [A2] to [A10], wherein the quantity ratio of thesurfactant A to the surfactant B (A/B) in the mixture (weight ratio) isnot less than 50/50 and less than 100/0.

[A12] The method for treating a sample containing blood componentsaccording to any one of [A1] to [A11], wherein the liquid containing thesurfactant A has a blood cell elimination effect as described in thefollowing (1);

(1) preparing a control sample by diluting a predetermined amount ofblood with distilled water so that an absorbance value measured by anabsorption photometry by use of a micro-plate reader and light of awavelength of not less than 600 nm is not less than 1 and less than 2;preparing a test sample by use of a liquid containing the surfactant Ain place of the distilled water in the control sample, so that the timefor the absorbance value of the test sample measured by an absorptionphotometry by use of light having a wavelength of not less than 600 nmis decreased to a half the absorbance value of the control sample is notmore than one hour.

[A13] The method for treating a sample containing blood componentsaccording to any one of [A1] to [A12], wherein the liquid containing thesurfactant A and the surfactant B, or the liquid containing thesurfactant A and the liquid containing the surfactant B or a mixture ofthe two liquids has the blood cell elimination effect of the following(2);

(2) preparing a control sample by diluting a predetermined amount ofblood with distilled water so that an absorbance value measured by anabsorption photometry by use of a micro-plate reader and light of awavelength of not less than 600 nm is not less than 1 and less than 2;preparing a test sample by use of a liquid containing the surfactant Aand the surfactant B, or a liquid containing the surfactant A and aliquid containing the surfactant B, or a mixture of these two liquids inplace of the distilled water in the control sample, so that the time forthe absorbance value of the test sample measured by an absorptionphotometry by use of light having a wavelength of not less than 600 nmis decreased to a half the absorbance value of the control sample notmore than 0.5 hours.

[A14] The method for treating a sample containing blood componentsaccording to any one of [A1] to [A13], wherein the method for treatingthe sample containing blood components is a method of lysingerythrocytes while suppressing degradation in the survival rate of rarecells that may be contained in the sample and/or degrading the survivalrate of leukocytes.

[A15] A method for separating or detecting a rare cell or a nucleic acidin a sample containing blood components, the method comprising treatinga sample containing blood components by any of the treatment methodsaccording to any one of [A1] to [A14], and separating or detecting therare cell or the nucleic acid from the treated sample.

[A16] A method for separating or detecting a rare cell or a nucleic acidin a sample containing blood components, comprising separating from asample containing blood components a fraction containing the rare cellor the nucleic acid, treating the separated fraction by the treatmentmethod according to any one of [A1] to [A14], and separating ordetecting the rare cell or the nucleic acid from the treated fraction.

[A17] The method for separating or detecting a rare cell or a nucleicacid in a sample containing blood components according to [A15] or[A16],

wherein the separation or the detection of the rare cell or the nucleicacid is carried out by a method selected from the group consisting ofseparation using affinity, density separation, centrifugationseparation, adhesion separation, sizing separation, flow cytometry,electrical separation, magnetic separation, sowing on a medium, and acombination thereof.

[A18] The method for separating or detecting a rare cell or a nucleicacid in a sample containing blood components according to any one of[A15] to [A17], wherein the rare cell is a cell selected from the groupconsisting of a cancer cell, circular tumor cell, a vascular endothelialcell, a vascular endothelial precursor cell, a cancer stem cell, anepithelial cell, a hematopoietic stem cell, a mesenchymal cell, a fetalcell, and a combination thereof.

[A19] The method for separating or detecting a rare cell or a nucleicacid in a sample containing blood components according to any one of[A15] to [A18], wherein the nucleic acid is a nucleic acid selected fromthe group consisting of: RNA and DNA in a blood component; RNA and DNAin a cancer cell, circular tumor cell, a vascular endothelial cell, avascular endothelial precursor cell, a cancer stem cell, an epithelialcell, a hematopoietic stem cell, a mesenchymal cell and a fetal cell;and a combination thereof.

[A20] The method for separating or detecting a rare cell or a nucleicacid in a sample containing blood components according to any one of[A15] to [A19], wherein the rare cell is labeled by use of a labelingmethod associated with a detection method selected from the groupconsisting of: a detection method using a radioactive substance, adetection method using a luminous phenomenon, a detection method using apigment, a detection method using magnetism, an electrical detectionmethod, an optical detection method, and a combination thereof.

[A21] A method for measuring the number of CTCs (circulating tumorcells) or a nucleic acid of a CTC in a sample containing bloodcomponents, the method comprising treating a sample containing bloodcomponents by the treatment method according to any one of [A1] to[A14], or separating/detecting a rare cell or a nucleic acid from asample containing blood components by the separation/detection methodaccording to any one of claims [A15] to [A20].

[A22] A method for assaying a rare cell in a sample containing bloodcomponents, the method comprising treating a sample containing bloodcomponents by the treatment method according to any one of [A1] to[A14], and subsequently assaying by a method comprising an observationof movements of the cell or an activity measurement.

[A23] A reagent kit comprising the surfactant A and the surfactant B tobe used for the treatment method according to any one of [A1] to [A14],the separation/detection method according to any one of [A15] to [A20],the CTC number measurement method according to [A21], and/or theassaying method according to [A22].

[A24] A reagent kit comprising the surfactant A to be used for thetreatment method according to any one of [A2] to [A14], theseparation/detection method according to any one of [A15] to [A20], theCTC number measurement method according to [A21], and/or the assayingmethod according to [A22].

EXAMPLES

The disclosure will be described below more specifically by referring tothe following Examples, though the Examples are not intended to limitthe disclosure.

1. Treatment with Polyoxyethylene Octyl Dodecylether (EO=25) (Examples1, 2)

Prepared was a 2.5 w/v % aqueous solution of polyoxyethylene octyldodecylether (EO=25) represented by Chemical formula (I) above (tradename: EMULGEN (trade name) 2025G, manufactured by Kao Corporation,Surfactant No. 1 in Table 1 below). The time for lysing erythrocytes atcontact with this aqueous solution was measured. Further, the survivalrate of leukocytes and the survival rate of cancer cells 10 minutesafter (Example 1) or 30 minutes after (Example 2) from the contact withthis aqueous solution were measured. Specifically, the measurements werecarried out under the conditions as stated below.

[Measurement of Lysis Time of Erythrocytes]

As a sample containing erythrocytes, blood that had been collected froma vein of a healthy person by use of a vacuum blood-collecting vessel(EDTA•including 2K) was used. 30 μL of the sample containing the bloodwas dispensed onto a 96-well microplate, in which 30 μL of the aqueoussolution of surfactant was mixed and stirred five times with pipetting,thereby allowing the reaction solution to react homogeneously. Theabsorbance was measured every one minute with a micro-plate reader. Themoment at which the absorbance became 0.5 or lower at a wavelength of650 nm was determined as the lysis of the blood cells. The results areshown in Table 3 below (Examples 1 and 2). In Examples 1 and 2, theabsorbance reached 0.5 within the time period from 30 minutes to 60minutes after starting the measurement. On the other hand, inComparative Example 9 where the aqueous solution of the surfactant wasreplaced by distilled water, the absorbance did never reach 0.5 even 60minutes after from the start of the measurement. The micro-plate readerused herein was Thermo Labsystems Multiskan Ascent 35 manufactured byThermo Labsystems.

[Assessment of Damage to Leukocytes]

Leukocytes were separated from 10 ml of fresh blood in accordance withHetaSep protocol (manufactured by STEMCELL (trade name) Technologies),which was suspended in 1 ml of PBS(−) and collected in a 1.5 mldisposable microtube (a microcentrifuge tube with trade name ofDSP-MC-15A manufactured by Nichiryo Co., Ltd.). Since a small amount oferythrocytes were still included in the separated leukocytes, theerythrocytes were eliminated by using ammonium chloride (supplied bySTEMCELL (trade name) Technologies) in accordance with the attacheddocument, and the leukocytes were collected through centrifugation,washed in PBS(−), and then suspended in 1 ml of a serum-includingmedium. 50 μL of leukocyte suspension in the serum-including medium and50 μL of the aqueous solution of the surfactant were mixed in the 1.5 mlmicrocentrifuge tube, and the mixed solution was stirred with pipettingabout five times so that the solution would be homogeneous.Subsequently, the solution was allowed to stand at an ambienttemperature of 23° C. for about 10 minutes to about 30 minutes so as toproceed the reaction. Later, the reaction liquid in this microcentrifugetube was stirred with pipetting so as to disperse the cells and then 50μL of the liquid was dispensed into another microcentrifuge tube inwhich 50 μL of 0.4% trypan blue stain solution (manufactured byInvitrogen) was mixed. This was injected into a counting chamber (CellCounter Plate: Advanced Neubauer type 177-112C manufactured by WATSONCo., Ltd.) so as to count living cells that had not been stained withthe trypan blue. Provided that the number of cells in a reaction liquidto which distilled water had been added in place of the reagent being100%, the survival rate was calculated from the number of cells survived10 minutes after (Example 1) and 30 minutes after (Example 2) from thereaction with the aqueous solution of surfactant. The results are shownin Table 3 below.

[Assessment of Damage to Cancer Cells]

Cells of SNU-1, MCF-7, SW620, PC3, and NCI-358 derived from gastriccancer, breast cancer, large intestinal cancer, prostate cancer, andlung cancer were prepared to be 2×10⁶ Cells/ml and used. The cancercells were prepared in the following manner. 50 μL of a cancer cellsuspension in the serum-including medium and 50 μL of the aqueoussolution of the surfactant were added into the 1.5 ml microcentrifugetube, stirred with pipetting so as to make the solution homogeneous.Later, the solution was allowed to stand at an ambient temperature of23° C. for about 10 minutes so as to proceed the reaction. Then, thereaction liquid in the microcentrifuge tube was stirred with pipettingso as to disperse the cells, and subsequently, 50 μL of the liquid wasdispensed into another microcentrifuge tube and 50 μL of 0.4% trypanblue stain solution (Invitrogen) was mixed therein. This was injectedinto a counting chamber so as to count living cells that had not beenstained with the trypan blue. Provided that the number of cells in areaction liquid to which distilled water had been added in place of thereagent being 100%, the survival rate was calculated from the number ofcells survived 10 minutes after (Example 1) and 30 minutes after(Example 2) from the reaction with the aqueous solution of thesurfactant. The results are shown in Table 3 below.

(Preparation of Cancer Cells)

According to an ordinary method, after eliminating the supernatants,media of human mammary epithelial cancer (cell strain name: MCF-7supplied by DS Pharma Biomedical Co., Ltd.), human colonic gland cancer(cell strain name: SW620 supplied by DS Pharma Biomedical Co., Ltd.),human prostate cancer (cell strain name: PC3 supplied by DS PharmaBiomedical Co., Ltd.) and human alveolar cell carcinoma (cell strainname: NCI-H358 supplied by DS Pharma Biomedical Co., Ltd.), which hadbeen cultivated on TC Dish (Greiner Bio-one), were washed with PBS(−) inorder to further eliminate the supernatants, and then was treated withtrypsin (supplied by Invitrogen) at 37° C. for 3 minutes. After adding aserum-including medium thereto, each of the cancer strains was collectedin a 15 ml centrifugation tube. This was centrifuged by using acentrifugal machine (CF20F supplied by Hitachi, Ltd.), therebyeliminating the supernatant. This was again suspended in theserum-including medium and collected. A human gastric cancer cell strain(SNU-1: ATCC) as a floating type was collected into a 15 mlcentrifugation tube without a trypsin treatment. After centrifugation, aserum-including medium was added before suspending and collecting thecell strain.

As indicated by Examples 1 and 2 in Table 3, the aqueous solution of thesurfactant No. 1 (polyoxyethylene octyl dodecylether (EO=25)) can lysethe erythrocytes and lower selectively the survival rate of theleukocytes rather than cancer cells. The effect became remarkable 30minutes after the contact with the surfactant No. 1.

2. Treatment Using Combination of Two Kinds of Surfactants (Examples3-7)

Using 14 kinds of surfactants (manufactured by Kao Corporation, NacalaiTesque Inc., Dojindo Molecular Technologies, Inc., and Lion Akzo Co.,Ltd.) recited in Table 1 below, mixtures were prepared by settingcombinations of Examples 3-7 and Comparative Examples 1-8 shown in Table3 below. Regarding the surfactant mixtures of Examples 3-7 andComparative Examples 1-8, the mixing ratio of the two surfactants(weight ratio) was set to 2:8 (i.e., the surfactant No. 1 occupies 80%).The total concentration of the surfactant in each of the surfactantmixtures was set to 2.5 w/v %. Comparative Example 9 indicates anexample where distilled water including no surfactant was used.Similarly to Example 1, the time for lysing erythrocytes at the time ofcontacting with any of these surfactants was measured, so as to measurethe survival rate of leukocytes and the survival rate of cancer cells 10minutes after the contact with the surfactant. The specific conditionswere similar to those of Example 1 except that the aqueous solution ofsurfactant in Example 1 was replaced by the mixture of surfactants. Theresults are shown in Table 3. Table 2 shows the measurement result forthe erythrocyte lysis time of a 2.5 w/v % aqueous solution for a casewhere each of the surfactants No. 1 to No. 6 was used alone.

TABLE 1 Surfactant Reagent name Hydrophobic No. (or trade name)Hydrophilic part group 1 EMULGEN 2025G Nonionic surfactant(polyoxyethylene alkyl ether), Double strands of manufactured by KaoCorporation chain length (C*12, C8) 2 EMULGEN MS-110 Nonionic surfactant(polyoxyethylene polyoxypropylene Chain length alkyl ether, HLB value =12.7, cloudy point(° C./2% (C12) aqueous solution) = 55), manufacturedby Kao Corporation 3 EMULGEN Nonionic surfactant (polyoxyethylene alkylether), Double strands of 2020G-HA manufactured by Kao Corporation chainlength (C12, C8) 4 EMULGEN LS-110 Nonionic surfactant (polyoxyethylenepolyoxypropylene Chain length alkyl ether, HLB value = 13.4, cloudypoint(° C./2% (C12) aqueous solution) = 73), manufactured by KaoCorporation 5 EMUNONE 1112 Nonionic surfactant (polyoxyethylene fattyacid ester), Chain length HLB = 13.7, manufactured by Kao Corporation(C12) 6 Sucrose laurate Nonionic surfactant (sugar chain), HLB16, Chainlength manufactured by Dojindo Molecular Technologies, Inc. (C12) 7NEOPELEX Anion (sulfate group), manufactured by Kao Chain lengthCorporation (C12) + benzene ring 8 Lauryl sodium Anion (surfactanthaving carboxylic acid at hydrophilic Chain length carboxylate part),manufactured by Nacalai Tesque Inc. (C12) 9 Lauryltrimethyl Cation,manufactured by Nacalai Tesque Inc. Chain length ammonium chloride (C12)10 PELEX OP-T Anion (sulfate group), manufactured by Kao Double strandsof Corporation chain length (C8, C8) 11 Decyltrimethyl Cation,manufactured by Lion Akzo Co., Ltd. Chain length ammonium chloride (C10)12 AMPHITOL 20BS Dipolar ion, manufactured by Kao Corporation Chainlength (C12) 13 AMPHITOL 20N Dipolar ion, manufactured by KaoCorporation Chain length (C12) 14 AMPHITOL 20YB Dipolar ion,manufactured by Kao Corporation Chain length (C12) *The number after ‘C’denotes the carbon number.

TABLE 2 Lysis time to reach Surfactant (reagent Preparation absorbanceof 0.5 No. name or trade name) concentration w/v % (min) 1 2025G 2.530-60 2 MS-110 2.5 1 3 2020HA 2.5 2 4 LS-110 2.5 1 5 EMUNONE 2.5 2 6Sucrose laurate 2.5 1 — Water — Not achieved

TABLE 3 Cancer cell survival rate (%)(10 minutes after a Surfactantcontact with surfacant) (2.5 w/v %) Lysis time SW620 mixing ratio toreach Leukocyte SNU-1 MCF-7 Large PC3-9 MCI-358 2:8 absorbance survivalGastric Breast intestinal Prostate Lung No. No. of 0.5 (min) rate (%)cancer cancer cancer cancer cancer Ex. 1 1 1 30-60 71.8 86.1 112.3 81101.8 89.9 Ex. 2 10.5* 60.9* 70.8* — — — Ex. 3 2 1 9 37 93.7 86.2 85.28106.9 85.1 Ex. 4 3 1 8 25.6 84.5 101.4 92.64 18.9 78.3 Ex. 5 4 1 6 7.760.5 65.0 5 8.0 37.8 Ex. 6 5 1 6 5.1 0.8 22.2 0 67.6 14.9 Ex. 7 6 1 2988.5 5.5 86.2 108 — 55.7 Co. Ex. 1 7 1 2 0 0.0 0 0 0.0 0 Co. Ex. 2 8 1 40.0 0.0 0.0 — 4.9 0.0 Co. Ex. 3 9 1 2 0 0.0 0.0 0 0.0 0.0 Co. Ex. 4 10 12 0 0.0 0 0 0.0 0 Co. Ex. 5 11 1 14 0 0.4 0.0 0 0.0 0.0 Co. Ex. 6 12 1 41.3 1.3 0.0 0 0.0 0.5 Co. Ex. 7 13 1 5 3.8 0.0 0.0 0 0.0 0.0 Co. Ex. 814 1 27 50.0 7.1 53.2 20 5.1 14.4 Co. Ex. 9 Distilled (Not 100 100.0 100100 100.0 100 water achieved) Note: Ex.: Example, Co. Ex.: ComparativeExample Example 2 refers to a result of measurement 30 minutes after acontact with surfactant. “—” indicates that no measurement was carriedout.

As shown in Table 3, in Examples 1 and 2 where the nonionic surfactantNo. 1 is used alone, lysis of the erythrocytes was achieved.Furthermore, in comparison with Comparative Examples 1-8, the survivalrate of cancer cells was kept high. In Examples 3-7 where the treatmentwas carried out with the mixture of surfactants No. 1 and any of Nos.2-6, the time for lysis of erythrocytes was shortened in comparison withExample 1, and a survival rate was kept high for at least one kind ofthe cancer cells in comparison with Comparative Examples 1-8. From theviewpoint of lowering the survival rate of leukocytes for the equal timeperiod (10 minutes), Examples 3-6 using respectively the surfactantsNos. 2-5 were superior to Examples 1 and 7. From the viewpoint ofkeeping the survival rate high for more kinds of cancer cells, Examples2-5 and 7 are superior to Example 6. Among them, Examples 3, 4 and 7, inparticular, Examples 3 and 4 were favorable.

3. Relationship Between Mixing Ratio of Two Kinds of Surfactants andDamage to Cancer Cell

A mixture of the surfactant No. 1 and either the surfactant No. 2 or No.3 was prepared at a mixing ratio of 10:0 to 0:10 (weight ratio: thetotal concentration of the surfactants was 2.5 w/v %). These mixtureswere used for assessing the damage to gastric cancer cells (SNU-1). Thedamage assessment was carried out as mentioned above. Namely, 50 μL of acancer cell suspension in the serum-including medium (2×10⁶ Cells/ml)and 50 μL of the mixture were mixed in a 1.5 ml microcentrifuge tube,stirred with pipetting about five times so as to make the solutionhomogeneous. Later, the solution was allowed to stand and react at anambient temperature of 23° C. for about 10 minutes. Then, the reactionliquid in the microcentrifuge tube was stirred with pipetting so as todisperse the cells, and subsequently, 50 μL of the liquid was dispensedinto another 1.5 ml microcentrifuge tube and mixed with 50 μL of 0.4%trypan blue stain solution (supplied by Invitrogen). This was injectedinto a counting chamber so as to count living cells that had not beenstained by the trypan blue, using an inverted microscope (supplied byOlympus Corporation). The survival rate was calculated from the numberof cells survived after the reaction with the mixture of surfactants,provided that the number of cells in a reaction liquid to whichdistilled water had been added in place of the reagent being 100%. Theresults with regard to the mixture the surfactant No. 2 and thesurfactant No. 1 are shown in FIG. 1 and the results with regard to themixture the surfactant No. 3 and the surfactant No. 1 are shown in FIG.2.

As shown in FIGS. 1 and 2, in a case where the mixing ratio of each ofthe surfactant No. 2 and No. 3 (weight ratio) exceeded 50%, the survivalrate of cancer cells was degraded considerably. In a case where themixing ratio of the surfactant No. 1 (weight ratio) exceeded 50%, thesurvival rate of cancer cells was raised considerably. In a case wherethe mixing ratio of the surfactant No. 1 (weight ratio) was 60% orhigher, the improvement of the survival rate of cancer cells wasremarkable.

4. Adhesion and Separation of Cancer Cells from a Sample ContainingBlood Containing a Cancer Cell

The cell surface of MCF-7 cell as a breast cancer cell was washed withPBS(−) (Dulbecco's PBS(−) manufactured by Nissui Pharmaceutical Co.,Ltd.) and then the cell was peeled off by trypsinization (37° C., 3minutes), and suspended in a serum-including medium. The cells werecounted with a counting chamber, and 1×10⁶ of cells were introduced intoeach centrifugal tube for centrifugation, from which supernatants wereeliminated and to which 1 ml of blood was added and suspended so as toprepare a sample containing blood containing a cancer cell. Into this 1ml of a sample containing blood containing a cancer cell, 1 ml ofsurfactant mixture of either Example 3 or 4 (mixture of the surfactantNo. 1 with either the surfactant No. 2 or the surfactant No. 3; thetotal concentration was 2.5 w/v %) was mixed, stirred five times withpipetting, and allowed to react for 10 minutes.

The blood cells are lysed during the reaction and the density of thereaction solution is increased. For this reason, at the time ofcentrifugation separation of the cells, an appropriate amount of bufferis added to lower the density. Specifically, 10 ml of reactant and thebuffer were introduced into a centrifugal tube and subjected tocentrifugation for 3 minutes at 1500 rpm and at ambient temperature (24°C.) in a centrifugal machine (CR20F manufactured by Hitachi, Ltd.).After eliminating the supernatant, the reactant is suspended in a mediumincluding 10 ml of serum, the cells are sown on a 10 cm TC Dish (GreinerBio-one) and kept in a 5% CO₂ incubator at 37° C. After 24 hours, themedium was exchanged, and the cells were kept again in the incubator.FIG. 3 includes photographs showing the growing conditions 60 hoursafter and 100 hours after. As shown in FIG. 3, cancer cells werecollected from the samples containing blood containing a cancer cell byuse of the surfactant mixture of Example 3 or 4, and the cancer cellsadhered to the cultivation dish and proliferated. Namely, it waspossible to further separate and/or concentrate the cancer cells afterthe reaction treatment and centrifugal separation. In a case where thecells were sown without the above-mentioned reaction treatment, theblood components such as blood cells and blood platelets hindered thecancer cells from adhering and growing.

5. Filtering of Blood Sample Containing Fluorescent Stained Cancer Cell

Cancer cells that had been fluorescent stained were mixed with blood soas to prepare a blood sample containing a cancer cell. This was treatedwith a surfactant mixture, and filtered so as to observe the cellscollected on the filter. Specific procedures are described below.

[Blood Sample Containing a Cancer Cell]

As a sample containing blood containing a cancer cell, 1 ml of blood towhich about 6000 cells of gastric cancer (SNU-1) stained previously inthe following manner was prepared.

(Staining of Cancer Cell)

The cancer cells (SNU-1) prepared similarly to the above [1.] werecollected from the culture solution, and then subjected tocentrifugation for 3 minutes in a centrifugal machine (CR20Fmanufactured by Hitachi, Ltd.) at 1500 rpm at ambient temperature (24°C.). After removing the supernatant, PBS(−) was added to suspend thecells. The cells suspended in the PBS(−) were fluorescent-stained. Forthe staining reagent, CellTracker (Invitrogen) was used. The CellTrackeris a reagent that can pass through even the cell membrane of a livingcell, and thus it reacts with the intracellular substance and istransformed to a fluorescent substance. The CellTracker was dissolved tobe 10 mM in DMSO and used such that the final concentration at thereaction would be in the range of 0.5 to 0.25 μM. It was allowed toreact in the PBS(−) for 30 to 40 minutes. The cancer cells (SNU-1) werestained with CellTracker Green (CMFDA/C7025) that emits greenfluorescence.

[Surfactant Mixture]

As the surfactant mixture, the surfactant mixture of Example 4 (mixtureof surfactant No. 3 and surfactant No. 1: the total concentration of thesurfactants was 2.5 w/v %) was prepared.

[Filter]

Nuclepore Track Etch Membrane of 013 mm and having a pore size of 8 μm(supplied by GE Healthcare) was fixed with a Swinnex Filter Holder(SX0001300 supplied by Yamato Scientific Co., Ltd.), thereby forming afilter. A plastic syringe from which a piston had been detached wasattached to the upper part of the filter holder. The lower part of thefilter was connected with a silicone tube, and the top end of the otherside was fixed inside a liquid waste bottle, thereby the filter wasused.

[Filtration]

In 1 ml of the sample containing the blood containing a cancer cell, 1ml of the surfactant mixture of Example 2 was mixed, stirred five timeswith pipetting and allowed to react for 10 minutes. The reactant wasadded to a syringe connected to the upper part of the filter forfiltration. Subsequently, a PBS(−) solution was added quietly before theblood passing through the filter entirely, thereby washing the filter.Later, fixation was carried out for about 30 minutes with a 4%paraformaldehyde/PBS buffer solution (Wako Pure Chemical IndustriesLtd.). The cells fixed on the membrane of the filter were observed witha microscope. The results are shown in FIG. 4B. FIG. 4A shows theresults of a case where blood was filtered without any treatment by useof such a surfactant mixture.

In FIG. 4B showing a case where treatment with a surfactant mixture wascarried out, substantially no blood cells are observed while many bloodcells are observed in FIG. 4A. The dots in FIG. 4B are pores of thefilter. The shining cells in FIG. 4B are cancer cells stained and mixedin the blood previously. It can be observed that even after a reactionwith the surfactant, the blood cells are destroyed and the cancer cellsare captured on the filter. This result shows that the purity of cancercells residing on the filter is increased and thus detection and thesubsequent analysis will be carried out easily.

The above data indicate that because of the treatment with thesurfactant mixture, the blood cells and the like are destroyed while therare cells (cancer cells) in the sample containing the blood is imposedless damage, and thus it is possible to carry out adhesion separationand cultivation with high purity, filter separation, fluorescentlabeling and the like. As a result, a precise assay of the collectedrare cells (cancer cells) will be possible.

6. Noise Elimination in Visualization of Cancer Cell in a Blood SampleContaining a Cancer Cell

A sample was prepared by mixing cancer cells that had beenfluorescent-stained and leukocytes that had been stained. After atreatment with a surfactant mixture, the sample was observed with afluorescence microscope. Specifically, the sample was prepared under theconditions as mentioned below.

[Preparation of a Blood Sample Containing a Cancer Cell]

Similarly to the above [5.], cancer cells (SNU-1) were prepared bystaining with CellTracker Green CMFDA/C7025 that emits greenfluorescence, and leukocytes were prepared by staining with CellTrackerBlue CMF2HC/C12881 that emits blue fluorescence. 200 μL ofserum-including medium including 6700 cancer cells and 1.4×10⁶ ofleukocytes was introduced into a microcentrifuge tube, thereby preparinga blood sample containing a cancer cell. This was dispensed into othermicrocentrifuge tubes so that the content each would be 30 μL.

[Treatment with Surfactant Mixture and Observation with FluorescenceMicroscope]

The blood sample containing a cancer cell was treated under theconditions as illustrated in Table 4 below (Examples 8-10 andComparative Example 10). The concentrations recited in the table are theconcentration during the preparation. At the time of reaction with acell suspension, equivalent 30 μL was added and mixed. The reactiontemperature was the ambient temperature of 23° C. After the reaction,640 μL of medium was added, and centrifugation was carried out for 3minutes at 2500 rpm with a centrifugal machine (CF15D manufacture byHitachi, Ltd.). It was suspended again in 600 μl of PBS(−) and thencentrifuged again by using the same centrifugal machine under the sameconditions. Subsequently, it was suspended in 100 μl of PBS(−), 50 μl ofwhich was dispensed onto a 384 plate. This was centrifuged for 5 minutesat 400×g with a plate centrifugal machine (M20484-A000) supplied byKubota Corporation, and observed with a fluorescence microscope underthe conditions as illustrated in Table 5 below. FIG. 5 shows the resultsof fluorescence microscope observation. Table 4 shows the results ofcounting the number of cells in FIG. 5.

TABLE 4 Microscopic observation result Reaction SNU-1 SNU-1 LeukocyteLeukocyte Surfactant Content time (min) (pieces) (%) (pieces) (%) Ex. 8No. 2 + No. 1 2.5 w/v % 10 40 69.0 57 0.76 (No. 2:No. 1 = 2:8) Ex. 9 No.3 + No. 1 2.5 w/v % 10 31 53.4 187 2.50 (No. 3:No. 1 = 2:8) Ex. 10 No. 15.0 w/v % 30 35 60.3 291 3.88 Co. Ex. 10 Distilled water — 10 58 1007491 100

TABLE 5 <Condition> Object lens: 10x Blue excitation - green observationUV excitation - blue observation EX (excitation) DM BA (fluorescence) UVset 365/±10 400 or more 470/±20 B set 475/±15 505 or more 525/±10Exposure time SNU-1 labeling 24 msec Leukocyte labeling 64 msec

As shown in FIG. 5 and Table 4, in Examples 8-10, the number ofleukocytes was decreased considerably after the treatment, and the noisein the microscopic observation was decreased remarkably in comparisonwith Comparative Example 10.

7. Detection and Separation by Flow Cytometry

Here, leukocytes isolated from 10 ml of blood similarly to the above[1.] were used. For the cancer cells, SNU-1 cells were used. Formeasurement, an automated cell counter MoxiZ by ORFLO, which uses theelectrical sensing zone method (Coulter Principle) was used. Theelectrical sensing zone method is a method including flowing electriccurrent through apertures, and measuring and detecting as a change inthe voltage pulse or the like the change in the electrical resistance inproportion to the volume of particles passing through the apertures.Volume distribution histogram is obtained by counting and processing thevoltage pulse height one by one. Thereby the size (volume) and number ofthe cells can be measured.

A mixture of the surfactant No. 2 and the surfactant No. 1 at a mixingratio of 2:8 (weight ratio, the total concentration of the surfactantwas 2.5 w/v %) was prepared. By using the surfactant mixture, particlesize distribution of gastric cancer cells (SNU-1) and that of leukocyteswere measured. 50 μL of a cancer cell suspension (1×10⁵ Cells/ml) orleukocytes (3×10⁵ Cells/ml) in a serum-including medium and 50 μL of thesurfactant mixture were mixed in a 1.5 ml microcentrifuge tube, stirredabout five times with pipetting so as to make the solution homogeneous.Later, the solution was allowed to stand at an ambient temperature of23° C. for about 10 minutes so as to proceed the reaction. After thereaction, the reaction solution in the microcentrifuge tube was stirredwith pipetting so as to disperse the cells, and 75 μL thereof was pouredinto a special cassette for MoxiZ, thereby measuring the particle sizedistribution (Example 11). In a comparative example, distilled water wasused in place of the surfactant mixture (Comparative Example 11).

Next, the cancer cells and the leukocytes were mixed to prepare a cellsample so that the number of cells would be equal to the number of theabove-mentioned number of the cells. The cell sample was allowed toreact with the surfactant mixture and the particle size distribution wasmeasured similarly to Example 11 (Example 12). In a comparative example,distilled water was used in place of the surfactant mixture (ComparativeExample 12).

The data obtained after the measurement can be outputted as a CSV file.Smoothing was carried out by a 7-point simple moving average regardingthe data obtained for the respective sizes. FIGS. 6A and 6B each shows aresult obtained by superimposing data of the respective particle sizedistributions of cancer cells and leukocytes. FIGS. 7A and 7B showparticle size distributions of suspensions of a mixture of the cancercells and leukocytes.

As shown in FIGS. 6 and 7, in Comparative Examples 11 and 12 (FIGS. 6Band 7B), the particle size distribution of the larger particles ofleukocytes and the particle size distribution of the smaller particlesof the cancer cells are overlapped with each other in the cell counter(“overlapped area” in FIG. 6B, and FIG. 7B). As a result, it isdifficult to isolate and divide the leukocytes and SNU-1, and manyleukocytes will be mixed in the collected SNU-1. On the other hand, inExample 11 (FIGS. 6A and 7A), the leukocytes in the overlapped area arelysed, and thus there exists no or a small overlapped area. As theoverlapped area disappears or decreases, purity of the cancer cells isimproved. For example, by collecting the cells indicated with a dottedline (5.5 (au)), which has a size of lysed leukocyte or larger, thedistribution of the leukocytes and the distribution of the cancer cellscan be separated. In this way, it is possible to decide the particlesize electrically so as to separate and detect cells of any arbitrarysize. Further, detection and separation become possible optically by useof fluorescent labeling and scattered light.

8. Effect of pH of Surfactant Mixture on Cancer Cells and LeukocytesSuspended in a Physiological Solution

A surfactant mixture containing the surfactant No. 2 and the surfactantNo. 1 at a mixing ratio of 2:8 (weight ratio; the total concentration ofthe surfactant is 2.5 w/v %) was prepared by using each PBS(−) solutionso that the pH would be in a range of 4 to 10. A damage assessment withrespect to gastric cancer cells (SNU-1) was carried out by using thesesurfactant mixtures.

The damage assessment was carried out as mentioned above except that thecells were suspended not in a serum-including medium but in aphysiological saline. Namely, 50 μL of a cancer cell suspension of thephysiological saline (2×10⁶ Cells/ml) and 50 μL of the mixture weremixed in a 1.5 ml microcentrifuge tube, stirred with pipetting aboutfive times so as to make the solution homogeneous. Later, the solutionwas allowed to stand and react at an ambient temperature of 23° C. forabout 10 minutes. Then, the reaction liquid in the microcentrifuge tubewas stirred with pipetting so as to disperse the cells, andsubsequently, 50 μL of the liquid was dispensed into another 1.5 mlmicrocentrifuge tube and mixed with 50 μL of 0.4% trypan blue stainsolution (supplied by Invitrogen). This was injected into a countingchamber so as to count living cells that had not been stained by thetrypan blue, using an inverted microscope (supplied by OlympusCorporation). The survival rate was calculated from the number of cellssurvived after the reaction with the surfactant mixtures, provided thatthe number of cells in a reaction liquid to which PBS(−) including nosurfactant had been added in place of the reagent being 100%.

Similarly, leukocytes were suspended in a physiological saline (8.2×10⁶Cells/ml) and allowed to react. The survival rate was calculated fromthe number of cells survived after the reaction with the surfactantmixtures, provided that the number of cells in a reaction liquid towhich PBS(−) including no surfactant had been added in place of thesurfactant mixture being 100%.

The results are shown in FIG. 8 and Table 6 below. As shown in FIG. 8and Table 6, from the viewpoint of raising the survival rate of thecancer cells and lowering the survival rate of leukocytes, it ispreferable that the pH of the surfactant mixture is closer to neutral.

TABLE 6 Survival rate (%) pH pH 4.0 pH 6.0 pH 7.4 pH 8.4 pH 10 SNU-138.6 93.4 96.3 51.7 50.9 Leukocyte 0 4.9 4.9 7.3 7.3

INDUSTRIAL APPLICABILITY

The disclosure is useful in the field of collection, measurement andassay of rare cells in blood, for example, in the scientific fields suchas medicine and pharmacy, and/or medical fields such as diagnosis andtherapy.

The invention may be embodied in other forms without departing from thespirit or essential characteristics thereof. The embodiments disclosedin this application are to be considered in all respects as illustrativeand not limiting. The scope of the invention is indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

What is claimed is:
 1. A method for treating a sample containing bloodcomponents, the method comprising mixing a sample containing bloodcomponents with a liquid containing a surfactant A and a liquidcontaining a surfactant B, or with a liquid containing the surfactant Aand the surfactant B, wherein the surfactant A is a nonionic surfactantrepresented by General formula (I) below, and the surfactant B has alytic property with respect to an erythrocyte higher than the lyticproperty of the surfactant A with respect to an erythrocyte:R¹—O-(EO)n-R²  (I) in the formula (I), R¹ is a hydrocarbon group havinga branched chain and having a carbon number in the range of 12 to 40, EOis an oxyethylene group, n is an average addition mole number of EO inthe range of 23 to 50, and R² is a hydrogen atom or a hydrocarbon grouphaving a carbon number in the range of 1 to
 3. 2. A method for treatinga sample containing blood components, the method comprising mixing asample containing blood components with a liquid containing a surfactantA, wherein no fixation agent is used, and the surfactant A is a nonionicsurfactant represented by General formula (I) below:R¹—O-(EO)n-R²  (I) in the formula (I), R¹ is a hydrocarbon group havinga branched chain and having a carbon number in the range of 12 to 40, EOis an oxyethylene group, n is an average addition mole number of EO inthe range of 23 to 50, and R² is a hydrogen atom or a hydrocarbon grouphaving a carbon number in the range of 1 to
 3. 3. The method fortreating a sample containing blood components according to claim 1,wherein the surfactant A is a polyoxyethylene alkylene ether (EO=23-35).4. The method for treating a sample containing blood componentsaccording to claim 1, wherein the surfactant B is a nonionic surfactantselected from the group consisting of a polyoxyethylene polyoxyalkylenealkylether, a polyoxyethylene alkylene ether, (EO=8-22), apolyoxyethylene fatty acid ester, a saccharose fatty acid ester, asorbitan fatty acid ester, and a combination thereof.
 5. The method fortreating a sample containing blood components according to claim 1,wherein the surfactant B is a nonionic surfactant represented by Generalformula (III) below:R³—O-(AO)m/(EO)n-R⁴  (III) wherein R³ is an alkyl group having a carbonnumber in the range of 1 to 24, R⁴ is a hydrogen atom or an alkyl grouphaving a carbon number in the range of 1 to 3, AO is an oxyalkylenegroup having a carbon number in the range of 3 to 6, EO is anoxyethylene group, m and n are average addition mole numbers of AO andEO respectively, where m is a number in the range of 1 to 100 and n is anumber in the range of 0 to 50, and “I” indicates that the AO group andthe EO group may be added at random or as a block regardless of theorder.
 6. The method for treating a sample containing blood componentsaccording to claim 1, wherein the surfactant B is a nonionic surfactantrepresented by General formula (IV) below:R⁵—O-(EO)n-R⁶  (IV) wherein R⁵ is a hydrocarbon group having a carbonnumber in the range of 12 to 40, EO is an oxyethylene group, n is anaverage addition mole number of EO in the range of 8 to 22, and R⁶ is ahydrogen atom or a hydrocarbon group having a carbon number in the rangeof 1 to
 3. 7. The method for treating a sample containing bloodcomponents according to claim 1, wherein the surfactant B is a nonionicsurfactant represented by General formula (VI) below:R⁷—COO-(EO)n-R⁸  (VI) wherein R⁷ is a hydrocarbon group having a carbonnumber in the range of 10 to 40, EO is an oxyethylene group, n is anaverage addition mole number of EO in the range of 6 to 160, and R⁸ is ahydrogen atom or a hydrocarbon group having a carbon number in the rangeof 1 to
 3. 8. The method for treating a sample containing bloodcomponents according to claim 1, wherein the surfactant B is asurfactant having a sugar moiety as a hydrophilic part and either afatty acid chain or an alkyl chain as a hydrophobic part.
 9. The methodfor treating a sample containing blood components according to claim 1,wherein the surfactant B is saccharose laurate.
 10. A method fortreating a sample containing blood components, the method comprisingmixing a sample containing blood components with a liquid containing asurfactant A and a surfactant B, or with a liquid containing thesurfactant A and a liquid containing the surfactant B, wherein thesurfactant A is a polyoxyetheylene octyl dodecyl ether (EO=23-50); thesurfactant B is a nonionic surfactant having a lytic property withrespect to an erythrocyte higher than the lytic property of thesurfactant A with respect to an erythrocyte, and selected from the groupconsisting of a polyoxyethylene polyoxypropylene alkylether, apolyoxyethylene octyl dodecyl ether (EO=8-22), a polyoxyethylene fattyacid ester, a saccharose fatty acid ester, a sorbitan fatty acid ester,and a combination thereof.
 11. The method for treating a samplecontaining blood components according to claim 1, wherein the quantityratio of the surfactant A to the surfactant B in the mixture (A/B)(weight ratio) is not less than 50/50 and less than 100/0.
 12. Themethod for treating a sample containing blood components according toclaim 2, wherein the liquid containing the surfactant A has a blood cellelimination effect as described in (1) below: (1) preparing a controlsample by diluting a predetermined amount of blood with distilled waterso that an absorbance value measured by an absorption photometry by useof a micro-plate reader and light of a wavelength of not less than 600nm is not less than 1 and is less than 2; preparing a test sample by useof a liquid containing 2.5 to 5 w/v % of the surfactant A in place ofthe distilled water in the control sample, so that the time for theabsorbance value of the test sample measured by an absorption photometryby use of light having a wavelength of not less than 600 nm is decreasedto a half the absorbance value of the control sample is not more thanone hour.
 13. The method for treating a sample containing bloodcomponents according to claim 1, wherein the liquid containing thesurfactant A and the surfactant B, or the liquid containing thesurfactant A and the liquid containing the surfactant B has the bloodcell elimination effect of (2) below: (2) preparing a control sample bydiluting a predetermined amount of blood with distilled water so that anabsorbance value measured by an absorption photometry by use of amicro-plate reader and light of a wavelength of not less than 600 nm isnot less than 1 and is less than 2; preparing a test sample by use of aliquid containing 2.5 to 5 w/v % of the surfactant A and the surfactantB in place of the distilled water in the control sample, so that thetime for the absorbance value of the test sample measured by anabsorption photometry by use of light having a wavelength of not lessthan 600 nm is decreased to a half the absorbance value of the controlsample is not more than 0.5 hours.
 14. The method for treating a samplecontaining blood components according to claim 1, wherein the method fortreating the sample containing blood components is a method of lysing anerythrocyte while suppressing degradation in the survival rate of a rarecell that may be contained in the sample and/or degrading the survivalrate of a leukocyte.
 15. A method for separating or detecting a rarecell or a nucleic acid in a sample containing blood components, themethod comprising treating a sample containing blood components by thetreatment method according to claim 1, and separating or detecting therare cell or the nucleic acid from the treated sample.
 16. The methodfor separating or detecting a rare cell or a nucleic acid in a samplecontaining blood components according to claim 15, wherein theseparation or the detection of the rare cell or the nucleic acid iscarried out by a method selected from the group consisting of separationusing affinity, density separation, centrifugation separation, adhesionseparation, sizing separation, flow cytometry, electrical separation,magnetic separation, sowing on a medium, and a combination thereof. 17.The method for separating or detecting a rare cell or a nucleic acid ina sample containing blood components according to claim 15, wherein therare cell is a cell selected from the group consisting of a cancer cell,a circular tumor cell, a vascular endothelial cell, a vascularendothelial precursor cell, a cancer stem cell, an epithelial cell, ahematopoietic stem cell, a mesenchymal cell, a fetal cell, and acombination thereof.
 18. The method for separating or detecting a rarecell or a nucleic acid in a sample containing blood components accordingto claim 15, wherein the nucleic acid is a nucleic acid selected fromthe group consisting of: RNA and DNA in a blood component; RNA and DNAin a cancer cell, a circular tumor cell, a vascular endothelial cell, avascular endothelial precursor cell, a cancer stem cell, an epithelialcell, a hematopoietic stem cell, a mesenchymal cell and a fetal cell;and a combination thereof.
 19. The method for separating or detecting arare cell or a nucleic acid in a sample containing blood componentsaccording to claim 18, wherein the rare cell is labeled by use of alabeling method associated with a detection method selected from thegroup consisting of: a detection method using a radioactive substance, adetection method using a luminous phenomenon, a detection method using apigment, a detection method using magnetism, an electrical detectionmethod, an optical detection method, and a combination thereof.
 20. Amethod for measuring the number of CTCs (circulating tumor cells) or anucleic acid of a CTC in a sample containing blood components, themethod comprising treating a sample containing blood components by thetreatment method according to claim
 1. 21. A method for assaying a rarecell in a sample containing blood components, the method comprisingtreating a sample containing blood components by the treatment methodaccording to claim 1, and subsequently assaying by a method comprisingan observation of movements of the cell or an activity measurement. 22.A reagent kit comprising the surfactant A and the surfactant B to beused for the treatment method according to claim
 1. 23. A reagent kitcomprising the surfactant A to be used for the treatment methodaccording to claim 2.